Methods and compositions for modulating immune tolerance

ABSTRACT

The instant invention provides methods and compositions for modulation of the immune system. Specifically, the present disclosure provides methods and compositions for increasing T cell mediated immune response useful in the treatment of cancer and chronic infection.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 14/313,481 filed 24 Jun. 2014 and U.S. patent application Ser.No. 11/992,880 filed 27 Sep. 2010, which is a Continuation ofInternational Application No. PCT/US2006/038195 having an internationalfiling date of 28 Sep. 2006, which claims benefit of U.S. ProvisionalApplication No. 60/722,675, filed 30 Sep. 2005, each of which areincorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

Research supporting this application was carried out by the UnitedStates of America as represented by the Secretary, Department of Healthand Human Services.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on 2 Jan. 2016, isnamed 1420378_178US52_Sequence_Listing_ST25.txt and is 26,455 bytes insize.

BACKGROUND OF THE INVENTION

T regulatory cells (Treg) or suppressor T cells are functionally definedas T cells that inhibit the immune response by influencing the activityof another cell. These cells comprise a small population of thymusderived CD4⁺ T cells. Despite there small population, these T regulatorycells have a large regulatory effect on the immune system. Overactivityof these T regulatory cells can contribute to the resistance of tumorsand infections to the immune system, where this resistance may take theform of, e.g., tolerance to the tumor, progressing lesions in cancer,and persistent bacterial and viral infections, see, e.g., Shimizu, etal. (2002) Nat. Immunol. 3:135-142; Shimizu, et al. (1999) J. Immunol.163:5211-5218; Antony and Restifo (2002) J. Immunotherapy 25:202-206;McGuirk and Mills (2002) Trends Immunol. 23:450-455; Tatsumi, et al.(2002) J. Exp. Med. 196:619-628; Jonuleit, et al. (2001) Trends Immunol.22:394-400.

Additionally, T regulatory cells mediate inflammatory and autoimmunedisorders. For example, CD25⁺CD4⁺ T regulatory cells play a role inpreventing, e.g., autoimmune gastritis, thyroiditis, insulin-dependentdiabetes melitus (IDDM), inflammatory bowel disorders (IBD),experimental autoimmune encephalomyelitis (EAE), food allergies, andgraft rejection. Conversely, impaired T regulatory cell activity canpromote autoimmune disorders, see, e.g., Wing, et al. (2003) Eur. J.Immunol. 33:579-587; Sakaguchi, et al. (2001) Immunol. Revs. 182:18-32;Sufi-Payer, et al. (1998) J. Immunol. 160:1212-1218; Shevach (2001) J.Exp. Med. 193:F41-F45; Read and Powrie (2001) Curr. Op. Immunol.13:644-649.

To function properly, the immune system must discriminate between selfand non-self. When self/non-self discrimination fails the immune systemdestroys cells and tissues of the body and as a result causes autoimmunediseases. Regulatory T cells actively suppress activation of the immunesystem and prevent pathological self-reactivity, i.e. autoimmunedisease. The critical role regulatory T cells play within the immunesystem is evidenced by the severe autoimmune syndrome that results froma genetic deficiency in regulatory T cells.

Moreover, large numbers of these regulatory cells have been found incancerous tissue. The presence of regulatory T cells at the site of atumor results in the creation of a favorable environment for tumorgrowth. Ideally, the presence and action of T regulatory cells at thesite of tumor growth would be reduced so that an immune response couldbe mounted to the growing tumor.

The molecular mechanism by which regulatory T cells exert theirsuppressor/regulatory activity has not been definitively characterizedand is the subject of intense research. In vitro experiments suggestthat suppressive mechanism requires cell-to-cell contact with the cellbeing suppressed. However, the immunosuppressive cytokines TGF-beta andinterleukin-10 (IL-10) have also been implicated in regulatory T cellfunction.

Despite numerous attempts and reports, immunotherapeutic interventiondoes not often generate long lasting effector T cell responses andtherapeutic immunity. T regulatory (Treg) cells play a central role inregulation of self-tolerance and control of responses to alloantigens.They comprise 5-10% of all CD4⁺ T cells, constitutively express the CD25and CTLA-4 markers and suppress the activation of effector CD4⁺ and CD8⁺cells and even dendritic cells. They have been implicated in widevariety of immunosuppressive responses, such as maternal tolerance tothe fetus, autoimmunity and tumor survival. Dysfunction or depletion ofTreg cells lead to spontaneous onset of various immune or autoimmunedisorders, such as organ-specific autoimmune diseases in mice ornickel-allergic responses in humans. As recently reported, two subsetsof Treg cells, natural and adaptive, differ in terms of specificity andeffector mechanisms (Bluestone et al. (2003) Nat Rev Immunol. 3:253-7).While the natural Treg cell subset develops in the thymus to preventpotentially pathological autoimmune reactions, the adaptive Treg cellsdevelop as result of activation of mature T cells under particularconditions of sub-optimal antigen exposure and co-stimulation (Bluestoneet al. supra). Treg-mediated suppressive activity has been shown to beboth cell-cell contact independent and dependent. For example,antigen-specific tumor infiltrating human CD4⁺CD25⁺ Treg cells requirescell contact and ligand-specific activation (via LAGE protein). Tregcells migration, particularly skin-homing, is controlled by chemokines,which signal via binding to differentially expressed chemokinereceptors, namely CCR4 and CCR8 (thus, these cells are attracted toMDC/CCL22, TARC/CCL17, I-309/CCL1 or viral chemokine vMIP-I and vMIP-III(Iellem et al. (2001) J Exp Med. 194:847-53). Chemokine receptors arealso differentially expressed on various immune cells. For example,cutaneous T cell lymphoma and adult T-cell leukemia/lymphoma (ATLL)cells over express CCR4, which was recently associated with unfavorableoutcome of the disease (Ishida et al. (2003) Clin Cancer Res.9:3625-34). In fact, expression of CCR4 was also associated with skinhoming of Tregs and the infiltration of Tregs at tumor site, including Bcell malignancies which produce CCL2 (Scrivner et al. (2003) LeukLymphoma. 44:383-9).

Due to the involvement of T regulatory cells in the progression of manydiseases and disorders, the need exists to be able to modulate theactivity of these T cells and, ultimately, the entire immune system, forthe treatment of these diseases and disorders.

SUMMARY OF THE INVENTION

The instant invention is based, at least in part, on the discovery thatfusion molecules comprising a chemokine fused to a toxin moiety,optionally including a linker, are capable of depleting T regulatorycells. These molecules are useful in the treatment of cancer, andautoimmunity, and further, can be used for efficient administration of avaccine, e.g., a cancer vaccine.

Accordingly, in one aspect, the instant invention provides a method forincreasing T cell mediated immune response in a subject by administeringto the subject a fusion molecule comprising a chemokine receptor ligandand a toxin moiety, thereby increasing the T cell mediated immuneresponse.

In one embodiment, the subject has cancer, e.g., a T cell malignancysuch as cutaneous T cell leukemia and myeloma. In another embodiment,the subject has a chronic infection, e.g., HIV infection, HCV infection,HBV infection and TB infection.

In another embodiment, the fusion molecule is administered near thelocation of a tumor.

In another embodiment, the chemokine receptor ligand is selected fromthe group consisting of a chemokine and a chemoattractant. In a relatedembodiment, the chemokine receptor ligand is a chemokine, e.g., MC148,TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1. In a related embodiment,the chemokine receptor ligand is specific for a chemokine receptorselected from the group consisting of CCR8, CCR4, CXCR1 CXCR4, CXCR2,CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9 and CCR10. In a specific embodiment, the chemokine receptor ligandis specific for a chemokine receptor selected from the group consistingof CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, ortoxic fragment thereof, e.g., eosinophil-derived RNase (EDN),Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin, or a fragmentthereof. In another embodiment, the toxin moiety is a non-proteinaceoustoxin, e.g., a maytansinoid, a calicheamicin or a taxane.

In specific embodiment, the fusion molecule is TARC-EDN, TARC-PE38,MC148-PE38 and MC148-EDN.

In another aspect, the invention provides a method of locally depletingT regulatory cells in a subject by locally administering to the subjecta fusion molecule comprising a chemokine receptor ligand and a toxinmoiety, thereby depleting the T regulatory cells in the area of theadministration of the fusion molecule.

In one embodiment, the subject has cancer, e.g., a T cell malignancysuch as cutaneous T cell leukemia and myeloma. In another embodiment,the subject has a chronic infection, e.g., HIV infection, HCV infection,HBV infection and TB infection.

In another embodiment, the fusion molecule is administered near thelocation of a tumor.

In another embodiment, the method involves administering the subject avaccine near the location of the administration of the fusion molecule.

In another embodiment, the chemokine receptor ligand is selected fromthe group consisting of a chemokine and a chemoattractant. In a relatedembodiment, the chemokine receptor ligand is a chemokine, e.g., MC148,TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1. In a related embodiment,the chemokine receptor ligand is specific for a chemokine receptorselected from the group consisting of CCR8, CCR4, CXCR1 CXCR4, CXCR2,CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9 and CCR10. In a specific embodiment, the chemokine receptor ligandis specific for a chemokine receptor selected from the group consistingof CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, ortoxic fragment thereof, e.g., eosinophil-derived RNase (EDN),Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin, or a fragmentthereof. In another embodiment, the toxin moiety is a non-proteinaceoustoxin, e.g., a maytansinoid, a calicheamicin or a taxane.

In specific embodiment, the fusion molecule is TARC-EDN, TARC-PE38,MC148-PE38 and MC148-EDN.

In another aspect, the instant invention provides a method of inhibitingimmune suppression in a subject by administering to the subject a fusionmolecule comprising a chemokine receptor ligand and a toxin moiety,thereby inhibiting immune suppression in the subject.

In one embodiment, the subject has cancer, e.g., a T cell malignancysuch as cutaneous T cell leukemia and myeloma. In another embodiment,the subject has a chronic infection, e.g., HIV infection, HCV infection,HBV infection and TB infection.

In another embodiment, the fusion molecule is administered near thelocation of a tumor.

In another embodiment, the method involves administering the subject avaccine near the location of the administration of the fusion molecule.

In another embodiment, the chemokine receptor ligand is selected fromthe group consisting of a chemokine and a chemoattractant. In a relatedembodiment, the chemokine receptor ligand is a chemokine, e.g., MC148,TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1. In a related embodiment,the chemokine receptor ligand is specific for a chemokine receptorselected from the group consisting of CCR8, CCR4, CXCR1 CXCR4, CXCR2,CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9 and CCR10. In a specific embodiment, the chemokine receptor ligandis specific for a chemokine receptor selected from the group consistingof CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, ortoxic fragment thereof, e.g., eosinophil-derived RNase (EDN),Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin, or a fragmentthereof. In another embodiment, the toxin moiety is a non-proteinaceoustoxin, e.g., a maytansinoid, a calicheamicin or a taxane.

In specific embodiment, the fusion molecule is TARC-EDN, TARC-PE38,MC148-PE38 and MC148-EDN.

In another aspect, the invention provides a method of modulating thesuppressive effect of CD4+CD25+ regulatory T cells of T effector cellsin a subject by administering to the subject a fusion moleculecomprising a chemokine receptor ligand and a toxin moiety, therebyinhibiting immune suppression in the subject.

In one embodiment, the subject has cancer, e.g., a T cell malignancysuch as cutaneous T cell leukemia and myeloma. In another embodiment,the subject has a chronic infection, e.g., HIV infection, HCV infection,HBV infection and TB infection.

In another embodiment, the fusion molecule is administered near thelocation of a tumor.

In another embodiment, the method involves administering the subject avaccine near the location of the administration of the fusion molecule.

In another embodiment, the chemokine receptor ligand is selected fromthe group consisting of a chemokine and a chemoattractant. In a relatedembodiment, the chemokine receptor ligand is a chemokine, e.g., MC148,TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1. In a related embodiment,the chemokine receptor ligand is specific for a chemokine receptorselected from the group consisting of CCR8, CCR4, CXCR1 CXCR4, CXCR2,CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9 and CCR10. In a specific embodiment, the chemokine receptor ligandis specific for a chemokine receptor selected from the group consistingof CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, ortoxic fragment thereof, e.g., eosinophil-derived RNase (EDN),Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin, or a fragmentthereof. In another embodiment, the toxin moiety is a non-proteinaceoustoxin, e.g., a maytansinoid, a calicheamicin or a taxane.

In specific embodiment, the fusion molecule is TARC-EDN, TARC-PE38,MC148-PE38 and MC148-EDN.

In another aspect, the invention provides a method of treating a subjecthaving a chronic infection by administering to the subject a fusionmolecule comprising a chemokine receptor ligand and a toxin moiety,thereby treating the chronic infection in the subject.

In one embodiment, the chronic infection is a bacterial infection, e.g.,tuberculosis infection. In another embodiment, the infection is a viralinfection, e.g., HIV infection, HCV infection, HBV infection. In anotherembodiment, the infection is a fungal infection.

In one embodiment, the fusion molecule is administered locally.

In another embodiment, the chemokine receptor ligand is selected fromthe group consisting of a chemokine and a chemoattractant. In a relatedembodiment, the chemokine receptor ligand is a chemokine, e.g., MC148,TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1. In a related embodiment,the chemokine receptor ligand is specific for a chemokine receptorselected from the group consisting of CCR8, CCR4, CXCR1 CXCR4, CXCR2,CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9 and CCR10. In a specific embodiment, the chemokine receptor ligandis specific for a chemokine receptor selected from the group consistingof CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, ortoxic fragment thereof, e.g., eosinophil-derived RNase (EDN),Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin, or a fragmentthereof. In another embodiment, the toxin moiety is a non-proteinaceoustoxin, e.g., a maytansinoid, a calicheamicin or a taxane.

In specific embodiment, the fusion molecule is TARC-EDN, TARC-PE38,MC148-PE38 and MC148-EDN.

In one embodiment, the invention provides a method of treating a subjecthaving cancer by administering to the subject a fusion moleculecomprising a chemokine receptor ligand and a toxin moiety, therebyinhibiting immune suppression in the subject.

In a related embodiment the cancer is a T cell malignancy, e.g.,cutaneous T cell leukemia or myeloma.

In one embodiment, the fusion molecule is administered near the locationof a tumor.

In one embodiment, the fusion molecule is administered locally.

In another embodiment, the chemokine receptor ligand is selected fromthe group consisting of a chemokine and a chemoattractant. In a relatedembodiment, the chemokine receptor ligand is a chemokine, e.g., MC148,TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1 In a related embodiment,the chemokine receptor ligand is specific for a chemokine receptorselected from the group consisting of CCR8, CCR4, CXCR1 CXCR4, CXCR2,CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9 and CCR10. In a specific embodiment, the chemokine receptor ligandis specific for a chemokine receptor selected from the group consistingof CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, ortoxic fragment thereof, e.g., eosinophil-derived RNase (EDN),Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin, or a fragmentthereof. In another embodiment, the toxin moiety is a non-proteinaceoustoxin, e.g., a maytansinoid, a calicheamicin or a taxane.

In specific embodiment, the fusion molecule is TARC-EDN, TARC-PE38,MC148-PE38 and MC148-EDN.

In another aspect, the invention provides a pharmaceutical compositioncomprising a fusion molecule comprising a chemokine receptor ligand anda toxin moiety and a pharmaceutically acceptable carrier.

In one embodiment, the chemokine receptor ligand is a chemokine or achemoattractant. In one embodiment, the chemokine receptor ligand is achemokine, e.g., MC148, TARC/CCL17, I-309/CCL1, MDC/CCL22, and vMIP-1 Ina related embodiment, the chemokine receptor ligand is specific for achemokine receptor selected from the group consisting of CCR8, CCR4,CXCR1 CXCR4, CXCR2, CXCR3, CXCR5, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9 and CCR10. In a specific embodiment, thechemokine receptor ligand is specific for a chemokine receptor selectedfrom the group consisting of CCR8 and CCR4.

In another embodiment, the toxin moiety is a proteinaceous toxin, orfragment thereof, e.g., eosinophil-derived RNase, Pseudomonas exotoxin,diphtheria toxin, or anthrax toxin. In a related embodiment, the toxinmoiety is a non-proteinaceous toxin, e.g., a maytansinoid, acalicheamicin or a taxane.

In one embodiment, the pharmaceutical composition is used for thetreatment of a T cell malignancy, e.g., cutaneous T cell leukemia andmyeloma.

In another embodiment, the pharmaceutical composition is used for thetreatment of a chronic infection, e.g., HIV infection, HCV infection,HBV infection or TB infection.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G are schematic representations of representative molecules ofthe invention. Genes for the mature form of viral chemokine MC148, orhuman TARC, or its mutant non-active form MC148D were fused in framewith various RNase moieties, such as EDN (MC148-EDN, TARC-EDN andMC148D-EDN, respectively), or ANG (TARC-ANG), or the toxin moiety, PE38(TARC-PE38). Control constructs expressed chemokine fusions with the VLchain of mouse plasmacytoma MOPC315 (MC148-VL315) or with tumor antigenOFA (TARC-OFA), respectively. Representative constructs containedoptional c-myc and His peptide tags to enable purification and detection(Tag), and an optional spacer peptide (SP) separating the chemokine fromthe antigen to enable proper folding of the protein.

FIGS. 2A-B depict graphs indicating that human T cell leukemia cells(CEM), but not MOLT cells express CCR4.

FIGS. 3A-B depict graphs indicating that human TARC-EDN binds to and isinternalized by cells expressing CCR4. FIG. 3A depicts 10⁵ cells treatedwith 25 μg/ml of protein (Control, EDN, or human TARC-EDN) for 1 hour at4° C. FIG. 3B depicts 10⁵ cells treated with 25 μg/ml of protein(Control, EDN, or human TARC-EDN) for 1 hour at 37° C.

FIG. 4 depicts a graph indicating that fusion molecules of the inventionpreferentially kill T regulatory cells that express CCR4.

FIG. 5 depicts a graph indicating that the molecules of the inventionrevert suppressive effects of T regulatory cells on CD8+ T cells. Thelevel of DNA synthesis was measured for CD8+ cells by BRDUincorporation. CD25+/CD4+ cells were treated with 1 μg/ml of the proteinof the invention for 2 days, and then mixed with CD8+ cells. Cellproliferation was stimulated by the addition of 1 μg/ml anti-CD3antibody for 3 days. The bars, from left to right, are: CD25+/CD4+alone; CD25−/CD4+ alone; CD8+ alone; CD8+ and CD25+/CD4+; PBS; humanTARC-EDN; human TARC-PE38.

FIGS. 6A-B depict the effect of human TARC-PE38 on the vialibility ofCEM and MOLT4 cells. FIG. 6A depicts the effects of human TARC-PE38 onthe viability of CEM (CCR4+) and MOLT4 (CCR4−) cells. FIG. 6B depictsthe effects of human TARC-PE38 and human TARC-OFA (control) on thevialibility of CEM (CCR4+) cells.

FIGS. 7A-B depict the effect of human TARC-PE38 and human TARC-OFA onCEM(CCR4+) tumors in NOD-SCID mice 7 days after single injection ofproteins. As is evident from the pictures, human TARC-PE38 significantlydecreases the size of the tumor, whereas the control human TARC-OFA hasno effect.

FIGS. 8A-E demonstrate that chemokine fusions retain properties of fusedmoieties. (A) Chemokines fused with EDN exhibit RNase activity(MC148-EDN and TARC-EDN) while MC148 fused to VL315 had no RNaseactivity. Shown, mean±SD, of the representative data from threeindependent experiments with similar results. (B) TARC-EDN induceschemotaxis of human monocytes. A chemotaxis assay was performed usingmicrochemotaxis chambers as described in Materials and Methods. Data areplotted ±SD; *, P<0.01 versus corresponding dose of EDN. (C) MC148-EDNbinds to the receptor positive HEK293/CCR8, but not to the receptornegative parental HEK-293 cells. Cells were incubated in the mediumcontaining either PBS (thin solid line), EDN (dashed line), or TARC-EDN(thick solid line) for 30 min at 4° C. (D) TARC-EDN (solid line) bindsto CEM cells and down-regulates surface expression of CCR4. Controls forbinding are PBS (interrupted line) and EDN (dotted line). Cells wereincubated with 50 μg/ml proteins for 30 min at either 4° C. or 37° C.The protein binding was detected with rabbit polyclonal anti-EDNantibody, followed by FITC-conjugated anti-rabbit IgG antibody.Expression of CCR4 was detected with FITC-conjugated anti-human CCR4antibody. Representative data from at least three independentexperiments with similar results. (E) CEM cells internalize TARC-PE38when incubated at 37° C., but not at 4° C. Cells were incubated with 25μg/ml of TARC-PE38 for 1 hour and stained with anti c-myc antibody(9E10) and FITC-conjugated secondary antibody. Cells were visualizedusing fluorescence microscopy.

FIGS. 9A-D demonstrate that EDN fused with chemokines induces celldeath. (A) Cytotoxicity of the MC148-EDN toward the CCR8 expressingHEK293/CCR8 cells after incubation for 7 days (chemotoxin amounts shownat μg/ml). Cell viability (Mean±SD) was measured using WST assay andresults are presented in percentage from the values of the PBS-treatedcells. * P<0.05 is for comparisons with the PBS group. (B) Killing ofCCR4 expressing CEM cells with TARC-EDN incubated for 5 and 8 days witheither 10 μg/ml TARC-EDN or EDN alone, or PBS. Cell death was evaluatedby propidium iodide staining and flow cytometry analysis. P-value is forcomparisons between the TARC-EDN and PBS groups. (C). Percent ofapoptotic cells (annexin positive) (D) and dead cells (double positivefor annexin and PI) after incubation with 10 μg/ml TARC-PE38 orTARC-OFA, or PBS for 1, 2 and 3 days.

FIGS. 10A-C demonstrate the specificity of TARC-PE38 killing. (A)TARC-PE38 kills CCR4⁺ CEM cells and does not affect viability of CCR4⁻MOLT-4 cells. Cells were incubated with TARC-PE38 (shown in μg/ml) fortwo days and cell viability (mean±SD) was measured using WST assay.Results are presented as a percentage of the PBS-treated cells. * P<0.05is for comparisons between CEM and MOLT-4 data at the indicatedconcentrations of TARC-PE38. (B) CEM cells are killed by only TARC-PE38,and not with TARC-OFA or TGFα-PE38. Protein concentrations shown areμg/ml. *, P<0.05 is for comparisons between the TARC-PE38 and TARC-OFAgroups. (C) Kinetics of killing of CEM cells incubated with 10 μg/mlTARC-PE38, or controls (TARC-OFA, TARC-EDN, EDN and PBS) for 1-3 days. *P<0.05 is for comparisons between the TARC-PE38 and TARC-OFA groups. Theviability was assessed using WST assay. PBS-treated cells at thecorresponding time point. Representative data of at least threeindependent experiments with comparable data.

FIGS. 11A-E demonstrate that TARC-PE38 kills CEM tumor established inNOD-scid mice. Mice were mock treated (PBS) or intratumorally injectedwith 25 μg TARC-PE38 (B, D, E) or TARC-OFA (A, C, E) for 5 days startingfrom day 12 post tumor challenge. TARC-PE38 injections are associatedwith local necrosis (B) and disappearance of the palpable tumor mass (B)and tumor cells as evaluated after H&E stain at day 27 post tumorchallenge (D, shown in 200× original magnification). (E) Tumor volumeafter treatments with TARC-PE38, or TARC-OFA or PBS. Tumors wereestablished in NOD-SCID mice by subcutaneous injection of 2×10⁷ CEMcells. * P<0.05 is for comparisons between the TARC-PE38 and TARC-OFAgroups. Representative data from two independent experiments withcomparable data with 8 mice per group.

FIGS. 12A-D demonstrate the mechanism of tumor escape: TARC-PE38 killsselectively CCR4-positive tumors, but does not prevent outgrowth ofresistant CCR4-negative escapees. Although TARC-PE38 injectionseliminated any palpable signs of a tumor, tumor eventually grew back anddid not respond to additional TARC-PE38 treatments. Tumor cells fromPBS-treated animals (PBS) and relapsed tumor cells from TARC-PE38treated animals (TARC-PE38) were cultured ex vivo in the presence ofvarying doses of TARC-PE38 (A). In parallel, parental CEM cells (ATCC)were also incubated with TARC-PE38. * P<0.01 is for comparisons with theTARC-PE38 treated parental CEM cells. To assess outgrowth ofCCR4-negative CEM cells, parental CEM cells were treated with PBS, or 10μg/ml TARC-OFA or TARC-PE-38 once (TARC-PE38 1×) or 5 times (TARC-OFA,TARC-PE38 5×) (B). Then cells were washed with PBS and culturingcontinued for several weeks more before staining for CCR4 expression (C)and (D) and testing for cell viability. * P<0.01 is for comparisons withthe TARC-OFA treated group. Representative data from at least twoindependent experiments with comparable data.

FIGS. 13 A-B demonstrate the efficacy of systemic therapy withchemotoxin. At Day 0, NOD/SCID mice were injected i.v. (tail vein) with1×106 CEM cells (n=6). Starting from Day 20, 10 μg of TARC-PE38 orCTACK-PE38 (control chemotoxin) in 100 μl PBS were injected I.V. once aday for 4 days. At Day 25, mice were culled and spleens were removed. A.Some splenocytes were stained with anti-human CD45-FITC Ab and wereanalyzed by FACS to evaluate the percentage of spleen-infiltrating tumorcells. B. Ten million splenocytes were cultured in RPMI supplementedwith 10% FBS for 10 days and, thereafter, were analyzed for human CD45and human CCR4.

FIGS. 14A-D depict the expression of CCR4 on Tregs in freshly isolatedhuman peripheral blood CD4⁺ T cells (A). CD4⁺ cells, negatively selectedfrom PBMCs, were stained for expression of CD25 and CCR4 and tested byFACS. Numbers indicate percentage of cells in the correspondingquadrant. CCR4⁺ and CCR4⁻ Tregs express FoxP3 and suppress CD8⁺ cellproliferation (B). Expression of FoxP3 and GAPDH tested by RT/PCR ontotal RNA extracted from FACS-sorted human peripheral blood CD4⁺ cellfractions. (C) Tregs regulate T cell proliferation. CFSE-stained CD8⁺cells were cultured with various subsets of CD4⁺ cells (indicated).X-axes indicate ratio of cells used starting from 5×10⁴ CD8⁺ cells mixedwith 1×10⁴ CD4⁺ cells. CD8⁺ cells were stimulated with equal numbers ofautologous APCs plus 0.5 μg/ml of soluble anti-CD3 Ab for 4 days. Cellswere stained with PE-conjugated anti-CD8 Ab and were analyzed by FACS.The results are expressed as percentage of proliferation of CD8⁺ cellscultured in the absence of CD4⁺ cells. Data represent mean±SD oftriplicates. *P<0.01, CD25⁺CCR4⁺ versus CD25⁻CCR4⁺ cells at thecorresponding ratio. (D) Phenotype of Tregs in human peripheral blood.CD25⁺CD4⁺ cells were purified and stained with Abs to correspondingsurface markers (indicated) by FACS. Numbers represent percentage ofcells in the corresponding quadrant. Shown, representative data from atleast three independent experiments with similar results.

FIGS. 15A-B depict CCR4⁻Tregs activity requires an initial activationthrough TCR. Freshly isolated CCR4⁺Tregs and CCR4⁻Tregs were tested forability to suppress proliferation of CFSE-labeled allogeneic CD8⁺ cellsin MLR with irradiated allogeneic APCs in the presence (A) or absence(B) of 0.5 μg/ml soluble anti-CD3 Ab. Shown as percentage ofproliferation of CD8⁺ cells cultured in the absence of Tregs,representative data (mean±SD of triplicates) from at least threeindependent experiments with similar results. *P<0.01 versus CD25⁻CD4⁺cells at the corresponding ratio.

FIG. 16A-F depict the mechanism of Treg suppression. Cell contact isrequired for Tregs suppression. Despite the fact that conditioned media(CM) from CCR4⁺Tregs incubated with CD8⁺ cells contains higher amountsof IL-10 and IL-5 (A), it does not affect proliferation of CD8⁺ cells(B). To study effects of secreted suppressive factors, titrated amountsof CM from the suppression assay with CCR4⁺Tregs (Med CD25⁺CCR4⁺) orCCR4⁻Tregs (Med CD25⁺CCR4⁻) were mixed with anti-CD3 Ab (0.5μg/ml)—stimulated CD8⁺ T cells. Shown as percentage of proliferation ofCD8⁺ T cells alone (mean±SD of triplicates). *^(†)P<0.01 for comparisonswith CD25⁺CCR4⁻ or CD25⁻CCR4⁺ groups, respectively. (C) CCR4⁻ but notCCR4⁺ cells express GZ-B after activation. Purified cells were activatedwith bead-bound antiCD3/CD28 Abs and were cultured for 5 days. IL-2 (10u/ml) was added to culture medium during the last 2 days of incubation.(D) CCR4+ but not CCR4-Tregs express CD95L/FasL. Purified cells wereincubated in RPMI medium supplemented with 10% FBS and 5% human AB serumfor 16 hours. Surface expression of CD95L/FasL and intracellularexpression of GZ-B were evaluated after staining with appropriate Abs byFACS. Histograms, thick line represents CD95L expression and thinline-isotype-matched control. Numbers represent percentage of cells inthe corresponding quadrant or gate. (E) CCR4⁻Tregs express CD95L/FASLafter activation. CCR4⁻Tregs were activated with bead-boundanti-CD3/CD28 Abs (activated) or cultured without stimuli(non-activated) for 16 hours. Cells were surface-stained with anti-CCR4and antiCD95L Abs. Numbers represent percentage of cells in thecorresponding quadrant. Representative data from two independentexperiments with similar results. (F) Suppression-induced with Tregs isdependent on CD95L/FasL. Subsets of Tregs or non-Treg CD25⁻CD4⁺ T cells(2.5×10⁴) were mixed with CFSE-labeled CD8⁺ T cells (5×10⁴) in four dayassay in the presence of anti-CD3 Ab (0.5 μg/ml) and autologous APCs.CD4⁺ cell subsets were pre-treated with 20 μg/ml anti-CD95L neutralizingor control Abs for 1 hour at 37° C. and then were mixed with CD8⁺ cellsand APCs. Shown as percentage of proliferation of CD8⁺ cells cultured inthe absence of Tregs (mean±SD of triplicates). *P<0.01 for comparisonswith control Ab treatment. Representative data from at least threeindependent experiments with similar results.

FIGS. 17A-E demonstrate that TARC-PE38 specifically kills purifiedCCR4⁺CD4⁺ cells (A). Control treatments were TARC-OFA and PBS. Shown aspercentage of viable cells to untreated cells evaluated by WST assayafter 2 days of treatment. Representative data (mean±SD of triplicates)from at least three independent experiments with similar results.*P<0.01 is for comparisons with control treatments. Depletion of CCR4⁺cells promotes CD4⁺ cell proliferation (B) and Th1 polarization (C).CD4⁺ T cells were overnight pretreated with 10 μg/ml TARC-PE38, orTARC-OFA, or PBS and were activated with soluble anti-CD3/anti-CD28 Abs.BrdU incorporation (B) and secreted IFNγ (C) were measured after sevendays of incubation. In parallel, proliferation and IFNγ secretion ofpurified CD4⁺CCR4⁺ and CD4⁺CCR4⁻ T cells was tested after the samestimulation. Depletion of the Th2-type non-Tregs with TARC-chemotoxin(TARC-PE38) reduces IL-10 (D) and augments IFNγ (E). CD4⁺ T cells werefirst depleted from CD25⁺ cells (Tregs) using anti-CD25 Ab-coatedmagnetic beads and the remaining cells were treated with TARC-PE38,TARC-OFA or PBS and stimulated with plate-bound anti-CD3 Ab. At theindicated time points, cytokines were measured by ELISA in conditionedmedia. Shown, representative (data mean±SD of triplicates) from at leasttwo independent experiments with similar results. *P<0.01 forcomparisons with PBS-treated groups.

FIGS. 18A-C demonstrate that the depletion of murine CCR4⁺Tregs augmentsmurine antigen specific T cell responses. (A) CD4⁺ T cells from C57BL/6mice were pretreated with TARC-PE38 or TARC-OFA, or PBS as in FIG. 4.Then cells were mixed with pmel CD8⁺ cells at 1:1 ratio and stimulatedfor five days with irradiated DCs from C57Bl/6 mice pulsed with 1 ng/mlof gp100₂₅₋₃₂ peptide. (B) Pmel splenocytes were pretreated withTARC-PE38, TARC-OFA or PBS as above and, then, cells were cultured forthree days with gp100₂₅₋₃₂ or control MOPC315 peptides. CD8⁺ cellproliferation was tested by BrdU incorporation. Data are shown aspercentage of proliferation of CD8⁺ cells alone. (C) IFNγ (μg/ml) wasmeasured in culture media of cells used in (B). Shown, representativedata (mean±SD of triplicates) from at least three independentexperiments with similar results.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention is based, at least in part, on the discovery thatfusion molecules comprising a chemokine fused to a toxin moiety,optionally including a linker, are capable of depleting T regulatorycells. These molecules are useful in the treatment of cancer, andautoimmunity, and further, can be used for efficient administration of avaccine, e.g., a cancer vaccine. Moreover, the instant inventionprovides methods and compositions which allow one of skill in the art touse a chemokine to preferentially kill cells expressing chemokinereceptor which binds the chemokine.

Accordingly, in one embodiment the invention provides methods ofcontrolling immune response.

Molecules of the Invention

The present invention provides fusion molecules, e.g., moleculescomprising a chemokine receptor ligand and one or more toxin moieties.The chemokine receptor ligand and the toxin moiety are optionally linkedby a linker, e.g., a peptide or non-peptide linker.

Exemplary chemokine receptor ligands include MC148 (viral chemokineencoded by the poxvirus molluscum contagiosum), TARC/CCL17 (thymus andactivation regulated chemokine), I-309/CCL1, MDC/CCL22, and vMIP-1(HHV-8 produced viral chemokine). Alternatively, chemokine receptorligands can be defined based on the receptor to which they bind. Forexample, chemokine receptor ligands used in the compositions and methodsof the invention may bind to a chemokine receptor such as CCR8 and CCR4.

One of skill in the art can identify other chemokine receptor ligandsand understands that homologues and orthologues of these molecules willbe useful in the methods of the instant invention. Moreover, variants ofthe chemokine receptor ligands are useful in the methods of theinvention.

The toxin moiety can be proteinaceous or non-proteinaceous. The term“non-proteinaceous” refers to toxin moieties that do not compriseprotein or protein-like moieties. In other words, a non-proteinaceoustoxin moiety is not peptide based and does not contain amino acids.Exemplary non-proteinaceous toxin moieties include maytansinoids (forexample, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0425 235 B 1), calicheamicins (for example, in U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296) or taxanes (such as paclitaxel and docetaxel).

The term “proteinaceous” as used herein refers to toxin moietiescomprising protein or protein-like moieties. Thus, the term encompassesmolecules that are naturally occurring proteins, fragments of naturallyoccurring protein toxins, and engineered polypeptides comprisingnaturally occurring amino acids, analogs of naturally occurring aminoacids, and combinations thereof. Exemplary proteinaceous toxin moietiesinclude eosinophil-derived RNase (EDN), Pseudomonas exotoxin, diphtheriatoxin, or anthrax toxin, or a toxic fragment thereof.

The fusion molecules of the invention may be assembledpost-translationally, i.e., the chemokine receptor ligand and the toxinmoiety can be covalently liked after being synthesized, or expressed,separately. Alternatively, the chemokine receptor ligand and toxinmoiety can be expressed as one transcript.

Nucleic Acid Molecules of the Invention

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid molecule encoding thefusion molecules, or components thereof, of the invention. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid molecule to which it has been linked.One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid molecule of the invention in a form suitable for expression of thenucleic acid molecule in a host cell, which means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which isoperatively linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein(e.g., fusion molecules comprising a chemokine receptor ligand and atoxin moiety).

The recombinant expression vectors of the invention can be designed forexpression of the polypeptides of the invention in prokaryotic oreukaryotic cells. For example, the polypeptides can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234), pMFa (Kudjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, the polypeptides can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banedji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Another aspect of the invention pertains to host cells into which anucleic acid molecule encoding a polypeptide of the invention isintroduced within a recombinant expression vector or a nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, apolypeptide of the invention can be expressed in bacterial cells such asE. coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding the polypeptide of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) the polypeptidesof the invention. Accordingly, the invention further provides methodsfor producing polypeptides using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector encoding apolypeptide of the invention has been introduced) in a suitable mediumsuch that a polypeptides of the invention is produced. In anotherembodiment, the method further comprises isolating the polypeptide fromthe medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichcoding sequences have been introduced. Such host cells can then be usedto create non-human transgenic animals in which exogenous sequences havebeen introduced into their genome or homologous recombinant animals inwhich endogenous sequences have been altered. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like.

Methods of Making the Molecules of the Invention

As described above, molecules of the invention may be made recombinantlyusing the nucleic acid molecules, vectors, and host cells describedabove.

Alternatively, the chemokine receptor ligand can be made synthetically,or isolated from a natural source and linked to the toxin moiety usingmethods and techniques well known to one of skill in the art.

Further, to increase the stability or half life of the fusion moleculesof the invention, the peptides may be made, e.g., synthetically orrecombinantly, to include one or more peptide analogs or mimmetics.Exemplary peptides can be synthesized to include D-isomers of thenaturally occurring amino acid residues to increase the half life of themolecule when administered to a subject.

Pharmaceutical Compositions

The nucleic acid and polypeptide fusion molecules (also referred toherein as “active compounds”) of the invention can be incorporated intopharmaceutical compositions. Such compositions typically include thenucleic acid molecule or protein, and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions of the instant invention may also includeone or more other active compounds. Alternatively, the pharmaceuticalcompositions of the invention may be administered with one or more otheractive compounds. Other active compounds that can be administered withthe pharmaceutical compounds of the invention, or formulated into thepharmaceutical compositions of the invention, include, for example,anticancer compounds, antiviral compounds, or antibacterial compounds.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Preferred pharmaceutical compositions of the invention are those thatallow for local delivery of the active ingredient, e.g., deliverydirectly to the location of a tumor. Although systemic administration isuseful in certain embodiments, local administration is preferred in mostembodiments.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack,kit or dispenser together with instructions, e.g., written instructions,for administration, particularly such instructions for use of the activeagent to treat against a disorder or disease as disclosed herein,including an autoimmune disease or disorder, treatment in connectionwith an organ or tissue transplant, as well as other diseases ordisorders with an autoimmune component such as AIDS. The container,pack, kit or dispenser may also contain, for example, a fusion molecule,a nucleic acid sequence encoding a fusion molecule, or a fusion moleculeexpressing cell.

Methods of Treatment

The compositions disclosed herein may be useful in the treatment ofcancer, chronic infection, i.e., viral or bacterial infection, and inthe administration of a vaccine to a subject.

The term “cancer” includes malignancies characterized by deregulated oruncontrolled cell growth, for instance carcinomas, sarcomas, leukemias,and lymphomas. The term “cancer” includes primary malignant tumors,e.g., those whose cells have not migrated to sites in the subject's bodyother than the site of the original tumor, and secondary malignanttumors, e.g., those arising from metastasis, the migration of tumorcells to secondary sites that are different from the site of theoriginal tumor.

The term “leukemia” includes malignancies of the hematopoietic cells ofthe bone marrow. Leukemias tend to proliferate as single cells. Examplesof leukemias include acute myeloid leukemia (AML), acute promyelocyticleukemia, chronic myelogenous leukemia, mixed-lineage leukemia, acutemonoblastic leukemia, acute lymphoblastic leukemia, acutenon-lymphoblastic leukemia, blastic mantle cell leukemia, myelodyplasticsyndrome, T cell leukemia, B cell leukemia, and chronic lymphocyticleukemia. Preferred leukemias include T cell malignancies, e.g., T cellleukemiz and myeloma.

The term “chronic infection” includes infections which may, for example,be bacterial infections (such as tuberculosis), parasitic infectionssuch as malarial infections or viral infections (such as HPV, HCV, HBVor HIV infections).

Examples of chronic infections associated include human hepatitisviruses such as hepatitis A, B, C, D and E, for example hepatitis Bvirus (HBV) and hepatitis C virus (HCV) which cause chronic hepatitis,cirrhosis and liver cancer (see U.S. Pat. No. 5,738,852).

Additional examples of chronic infections caused by viral infectiousagents include those caused by the human retroviruses: humanimmunodeficiency viruses (HIV-1 and HIV-2), which cause acquired immunedeficiency syndrome (AIDS); and human T lymphotropic viruses (HTLV-1 andHTLV-2) which cause T cell leukemia and myelopathies. Many otherinfections such as human herpes viruses including the herpes simplexvirus (HSV) types 1 and 2, Epstein Barr virus (EBV), cytomegalovirus(CMV), varicella-zoster virus (VZV) and human herpes virus 6 (HHV-6) areoften not eradicated by host mechanisms, but rather become chronic andin this state may cause disease. Chronic infection with human papillomaviruses is associated with cervical carcinoma. Numerous other virusesand other infectious agents replicate intracellularly and may becomechronic when host defense mechanisms fail to eliminate them. Theseinclude pathogenic protozoa (e.g., Pneumocystis carinii, Trypanosoma,Leishmania, Plasmodium (responsible for Malaria) and Toxoplasma gondii),bacteria (e.g., mycobacteria (eg Mycobacterium tuberculosis responsiblefor tuberculosis), salmonella and listeria), and fungi (e.g., candidaand aspergillus).

The invention provides therapeutic methods and compositions for theprevention and treatment of cancer and chronic infection and for theadministration of a vaccine to a subject. In particular, the inventionprovides methods and compositions for the prevention and treatment ofcancer and chronic infection in subjects as well as methods andcompositions that allow for efficient administration of a vaccine inhumans as well as other animals through the administration of fusionmolecules comprising a chemokine and a toxin moiety.

In one embodiment, the present invention contemplates a method oftreatment, comprising: a) providing, i.e., administering: i) a mammalianpatient particularly human who has, or is at risk of developing, canceror a chronic infection, ii) one or more fusion molecules of theinvention.

The term “at risk for developing” is herein defined as individuals withfamilial incidence of, for example, cancer or chronic infection.

The present invention is also not limited by the degree of benefitachieved by the administration of the fusion molecule. For example, thepresent invention is not limited to circumstances where all symptoms areeliminated. In one embodiment, administering a fusion molecule reducesthe number or severity of symptoms of cancer or a chronic infection. Inanother embodiment, administering of a fusion molecule may delay theonset of symptoms.

As mentioned above, the indications for which the administration offusion molecule compositions can be used include in particular cancer,e.g., T-cell related cancers, or chronic infection, e.g., HIV infection,HCV infection, HBV infection, TB infection.

Typical subjects for treatment in accordance with the individualsinclude mammals, such as primates, preferably humans. Cells treated inaccordance with the invention also preferably are mammalian,particularly primate, especially human. As discussed above, a subject orcells are suitably identified as in needed of treatment, and theidentified cells or subject are then selected for treatment andadministered one or more of fusion molecules of the invention.

The treatment methods and compositions of the invention also will beuseful for treatment of mammals other than humans, including forveterinary applications such as to treat horses and livestock e.g.cattle, sheep, cows, goats, swine and the like, and pets such as dogsand cats.

For diagnostic or research applications, a wide variety of mammals willbe suitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids (e.g., blood, plasma, serum, cellularinterstitial fluid, saliva, feces and urine) and cell and tissue samplesof the above subjects will be suitable for use.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Example 1

Materials and Methods

Fusion Gene Cloning and Plasmid Construction

Human and mouse TARC/CCL17 DNAs (mTARC and hTARC) were cloned from humanand mouse thymus cDNA library by PCR truncated Pseudomonas aeriginosaDNA fragment, PE38, and EDN DNA fragment. mTARC and hTARC or viral MC148chemokines were genetically fused in frame with toxin moieties (EDN orPE38), or with irrelevant tumor antigen OFA. Some constructs contained 6His (SEQ ID NO: 25) and c-myc tag at COO-end for protein purificationand analytical purposes. Chemokines were linked with toxin moieties viaflexible linker peptide sequence (SP). All constructs were verified byDNA sequencing. The sequence of exemplary chemotoxin fusions consistingof a chemokine, or chemo-attractant fused in frame with toxic moiety,such as EDN or PE38 are set forth below.

The nucleic acid sequence of TARC-PE38 chemotoxin is set forth as SEQ IDNO:1.

SEQ ID NO: 1 ATGGCACGAGGGACCAACGTGGGCCGGGAGTGCTGCCTGGAGTACTTCAAGGGAGCCATTCCCCTTAGAAAGCTGAAGACGTGGTACCAGACATCTGAGGACTGCTCCAGGGATGCCATCGTTTTTGTAACTGTGCAGGGCAGGGCCATCTGTTCGGACCCCAACAACAAGAGAGTGAAGAATGCAGTTAAATACCTGCAAAGCCTTGAGAGGTCTGATGGTGGTGGCTCTGGCGGTGGGGGTAGCCTCGAGGGTGGTGGTGGTTCTAAACCGCCGCAGTTCACTTGGGCTCAGTGGTTCGAAACTCAGCATATCAACATGACTTCTCAGCAGTGCACTAACGCTATGCAGGTTATCAACAACTACCAGCGTCGTTGCAAAAACCAGAACACTTTCCTGCTGACTACTTTCGCTAACGTTGTTAACGTTTGCGGTAACCCGAACATGACTTGCCCGTCTAACAAAACTCGTAAAAACTGCCATCATTCTGGTTCTCAGGTTCCGCTGATCCATTGCAACCTGACTACTCCGTCTCCGCAGAACATCTCTAACTGCCGTTACGCTCAGACTCCGGCTAACATGTTCTACATCGTTGCTTGCGACAACCGTGACCAGCGTCGTGACCCGCCGCAGTACCCGGTTGTTCCGGTTCATCTGGACCGTATCATCGGATCCGCAGAAGAACAGAAACTGATCTCAGAAGAGGATCTGGCCCACCACCATCACCATCACTAA

The Amino acid sequence of TARC-PE38 chemotoxin is set forth as SEQ IDNO:2. SEQ ID NO:2 is encoded by the nucleic acid molecule set forth asSEQ ID NO:1.

SEQ ID NO: 2 MARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPNNKRVKNAVKYLQSLERSDGGGSGGGGSLEGGGGSKPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRIIGSAEEQKLISEEDLAHHHHHH

The nucleic acid sequence of TARC-PE38 chemotoxin is set forth as SEQ IDNO:3.

SEQ ID NO: 3 ATGGCACGAGGGACCAACGTGGGCCGGGAGTGCTGCCTGGAGTACTTCAAGGGAGCCATTCCCCTTAGAAAGCTGAAGACGTGGTACCAGACATCTGAGGACTGCTCCAGGGATGCCATCGTTTTTGTAACTGTGCAGGGCAGGGCCATCTGTTCGGACCCCAACAACAAGAGAGTGAAGAATGCAGTTAAATACCTGCAAAGCCTTGAGAGGTCTGATGGTGGTGGCTCTGGCGGTGGGGGTAGCCTCGAAGCTTCTGGAGGTCCCGAGGGCGGCAGCCTGGCCGCGCTGACCGCGCACCAGGCTTGCCACCTGCCGCTGGAGACTTTCACCCGTCATCGCCAGCCGCGCGGCTGGGAACAACTGGAGCAGTGCGGCTATCCGGTGCAGCGGCTGGTCGCCCTCTACCTGGCGGCGCGGCTGTCGTGGAACCAGGTCGACCAGGTGATCCGCAACGCCCTGGCCAGCCCCGGCAGCGGCGGCGACCTGGGCGAAGCGATCCGCGAGCAGCCGGAGCAAGCCCGTCTGGCCCTGACCCTGGCCGCCGCCGAGAGCGAGCGCTTCGTCCGGCAGGGCACCGGCAACGACGAGGCCGGCGCGGCCAACGGCCCGGCGGACAGCGGCGACGCCCTGCTGGAGCGCAACTATCCCACTGGCGCGGAGTTCCTCGGCGACGGCGGCGACGTCAGCTTCAGCACCCGCGGCACGCAGAACTGGACGGTGGAGCGGCTGCTCCAGGCGCACCGCCAACTGGAGGAGCGCGGCTATGTGTTCGTCGGCTACCACGGCACCTTCCTCGAAGCGGCGCAAAGCATCGTCTTCGGCGGGGTGCGCGCGCGCAGCCAGGACCTCGACGCGATCTGGCGCGGTTTCTATATCGCCGGCGATCCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCGACGCACGCGGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGCTCGAGCCTGCCGGGCTTCTACCGCACCAGCCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCGAACGGCTGATCGGCCATCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCCGAGGAGGAAGGCGGGCGCCTGGAGACCATTCTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTCCCTCGGCGATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCGACCCGTCCAGCATCCCCGACAAGGAACAGGCGATCAGCGCCCTGCCGGACTACGCCAGCCAGCCCGGCAAACCGCCGCGCGAGGACCTGAAGAGATCCGCAGAAGAACAGAAACTGATCTCAGAAGAGGATCTGGCC CACCACCATCACCATCACTAA

Amino acid sequence of TARC-PE38 chemotoxin is set forth as SEQ ID NO:4.SEQ ID NO:4 is encoded by the nucleic acid molecule set forth as SEQ IDNO:3.

SEQ ID NO: 4 MARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPNNKRVKNAVKYLQSLERSDGGGSGGGGSLEASGGPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKRSAEEQKLISEEDLA HHHHHH

The nucleic acid sequence of I-309-EDN is set forth as SEQ ID NO:5.

SEQ ID NO: 5 ATGGCATCCATGCAGGTACCGTTCTCCCGCTGTTGCTTCTCATTTGCGGAGCAAGAGATTCCCCTGAGGGCAATCCTGTGTTACAGAAATACCAGCTCCATCTGCTCCAATGAGGGCTTAATATTCAAGCTGAAGAGAGGCAAAGAGGCCTGCGCCTTGGACACAGTTGGATGGGTTCAGAGGCACAGAAAAATGCTGAGGCACTGCCCGTCAAAAAGAAAATCCGGTGGTGGTGGTTCTGGCCTCGAGGGTGGTGGTGGTTCTAAACCGCCGCAGTTCACTTGGGCTCAGTGGTTCGAAACTCAGCATATCAACATGACTTCTCAGCAGTGCACTAACGCTATGCAGGTTATCAACAACTACCAGCGTCGTTGCAAAAACCAGAACACTTTCCTGCTGACTACTTTCGCTAACGTTGTTAACGTTTGCGGTAACCCGAACATGACTTGCCCGTCTAACAAAACTCGTAAAAACTGCCATCATTCTGGTTCTCAGGTTCCGCTGATCCATTGCAACCTGACTACTCCGTCTCCGCAGAACATCTCTAACTGCCGTTACGCTCAGACTCCGGCTAACATGTTCTACATCGTTGCTTGCGACAACCGTGACCAGCGTCGTGACCCGCCGCAGTACCCGGTTGTTCCGGTTCATCTGGACCGTATCATCGGATCCGCAGAAGAACAGAAACTGATCTCAGAAGAGGATCTGGCCCACCACCATCACCATCACTAA

The amino acid sequence of I-309-EDN is set forth as SEQ ID NO:6. SEQ IDNO:6 is encoded by the nucleic acid molecule set forth as SEQ ID NO:5.

SEQ ID NO: 6 MASMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRHRKMLRHCPSKRKSGGGGSGLEGGGGSKPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRIIGSAEEQKLISEEDLAHHHHHH

The nucleic acid sequence of MC148-EDN is set forth as SEQ ID NO:7.

SEQ ID NO: 7 ATGGCACTCGCGAGACGGAAATGTTGTTTGAATCCCACAAATCGTCCGATCCCGAATCCTTTACTGCAAGATCTATCACGCGTCGACTATCAGGCGATAGGACATGACTGCGGACGGGAAGCTTTCAGAGTGACGCTGCAAGACGGAAGACAAGGCTGCGTTAGCGTTGGTAACAAGAGCTTACTAGACTGGCTTCGGGGACACAAGGATCTCTGCCCTCAGATATGGTCCGGGTGCGAGTCTCTGGAATTCAACGACGCTCAGGCGCCGAAGAGTCTCGAGGGTGGTGGTGGTTCTAAACCGCCGCAGTTCACTTGGGCTCAGTGGTTCGAAACTCAGCATATCAACATGACTTCTCAGCAGTGCACTAACGCTATGCAGGTTATCAACAACTACCAGCGTCGTTGCAAAAACCAGAACACTTTCCTGCTGACTACTTTCGCTAACGTTGTTAACGTTTGCGGTAACCCGAACATGACTTGCCCGTCTAACAAAACTCGTAAAAACTGCCATCATTCTGGTTCTCAGGTTCCGCTGATCCATTGCAACCTGACTACTCCGTCTCCGCAGAACATCTCTAACTGCCGTTACGCTCAGACTCCGGCTAACATGTTCTACATCGTTGCTTGCGACAACCGTGACCAGCGTCGTGACCCGCCGCAGTACCCGGTTGTTCCGGTTCATCTGGACCGTATCATCGGATCCGCAGAAGAACAGAAACTGATCTCAGAAGAGGATCTGGCCCACCAC CATCACCATCACTAA

The amino acid sequence of MC148-EDN is set forth as SEQ ID NO:8. SEQ IDNO:8 is encoded by the nucleic acid molecule set forth as SEQ ID NO:7.

SEQ ID NO: 8 MALARRKCCLNPTNRPIPNPLLQDLSRVDYQAIGHDCGREAFRVTLQDGRQGCVSVGNKSLLDWLRGHKDLCPQIWSGCESLEFNDAQAPKSLEGGGGSKPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRIIGSAEEQKLISEEDLAHH HHHH

The nucleic acid sequence of MC148-PE38 is set forth as SEQ ID NO:9.

SEQ ID NO: 9 ATGGCACTCGCGAGACGGAAATGTTGTTTGAATCCCACAAATCGTCCGATCCCGAATCCTTTACTGCAAGATCTATCACGCGTCGACTATCAGGCGATAGGACATGACTGCGGACGGGAAGCTTTCAGAGTGACGCTGCAAGACGGAAGACAAGGCTGCGTTAGCGTTGGTAACAAGAGCTTACTAGACTGGCTTCGGGGACACAAGGATCTCTGCCCTCAGATATGGTCCGGGTGCGAGTCTCTGGAATTCAACGACGCTCAGGCGCCGAAGAGTCTCGAAGCTTCTGGAGGTCCCGAGGGCGGCAGCCTGGCCGCGCTGACCGCGCACCAGGCTTGCCACCTGCCGCTGGAGACTTTCACCCGTCATCGCCAGCCGCGCGGCTGGGAACAACTGGAGCAGTGCGGCTATCCGGTGCAGCGGCTGGTCGCCCTCTACCTGGCGGCGCGGCTGTCGTGGAACCAGGTCGACCAGGTGATCCGCAACGCCCTGGCCAGCCCCGGCAGCGGCGGCGACCTGGGCGAAGCGATCCGCGAGCAGCCGGAGCAAGCCCGTCTGGCCCTGACCCTGGCCGCCGCCGAGAGCGAGCGCTTCGTCCGGCAGGGCACCGGCAACGACGAGGCCGGCGCGGCCAACGGCCCGGCGGACAGCGGCGACGCCCTGCTGGAGCGCAACTATCCCACTGGCGCGGAGTTCCTCGGCGACGGCGGCGACGTCAGCTTCAGCACCCGCGGCACGCAGAACTGGACGGTGGAGCGGCTGCTCCAGGCGCACCGCCAACTGGAGGAGCGCGGCTATGTGTTCGTCGGCTACCACGGCACCTTCCTCGAAGCGGCGCAAAGCATCGTCTTCGGCGGGGTGCGCGCGCGCAGCCAGGACCTCGACGCGATCTGGCGCGGTTTCTATATCGCCGGCGATCCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCGACGCACGCGGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGCTCGAGCCTGCCGGGCTTCTACCGCACCAGCCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCGAACGGCTGATCGGCCATCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCCGAGGAGGAAGGCGGGCGCCTGGAGACCATTCTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTCCCTCGGCGATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCGACCCGTCCAGCATCCCCGACAAGGAACAGGCGATCAGCGCCCTGCCGGACTACGCCAGCCAGCCCGGCAAACCGCCGCGCGAGGACCTGAAGAGATCCGCAGAAGAACAGAAACTGATCTCAGAAGAGGATCTGGCCCACCACCATCACCATCACTA A

The amino acid sequence of MC148-PE38 is set forth as SEQ ID NO:10. SEQID NO:10 is encoded by the nucleic acid molecule set forth as SEQ IDNO:9.

SEQ ID NO: 10 MALARRKCCLNPTNRPIPNPLLQDLSRVDYQAIGHDCGREAFRVTLQDGRQGCVSVGNKSLLDWLRGHKDLCPQIWSGCESLEFNDAQAPKSLEASGGPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKRSAEE QKLISEEDLAHHHHHHRecombinant Fusion Proteins and Peptides

Fusion proteins were expressed in E. coli and purified from inclusionbodies with subsequent denaturation and refolding steps and affinitypurifications using FPLC chromatography. The integrity and purity (>90%)of recombinant proteins were tested by SDS-PAGE under reducingconditions and Western blot hybridization with 9E10 anti-c-myc mAb oranti-EDN antibody.

Cell Lines and Animals

Acute T-lymphoblastic leukemia CEM and MOLT-4 cells were purchased fromATCC and were cultured in RPMI medium supplemented with 10% FBS. Cellviability was determined by Annexin/PI staining or WST reagent. Cellproliferation was determined by BrDU incorporation.

Animals and in vivo Studies

NOD/SCID mice were purchased from Jackson (The Jackson Laboratory, BarHarbor, Me.). All animals were housed at the National Institute of Aginganimal facility, Baltimore, Md. Animal care was provided in accordancewith the procedures outlined in a Guide for the Care and Use ofLaboratory Animals. Six- to eight-weeks old female NOD/SCID mice wereinoculated with 20 million CEM cells subcutaneously. Two weeks aftertumor inoculation, proteins (25 μg) were injected into the establishedtumors.

Isolation of Human Peripheral Blood Mononuclear Cells (PBMC) and T CellSubsets

PBMCs were isolated from blood of healthy donors by Ficol densitygradient centrifugation. Human CD8+ and CD4+ T cells were isolated fromPBMCs using corresponding kits from R&D. CD4+/CD25+/CD4+ cells (Tregulatory cells) were isolated from human T cells using MACS kit fromMiltenyi Biotec.

Chemokine Receptor Expression and Binding

The ligand binding-internalization assays were performed with CEM orMOLT-4 cells (1×10⁶) blocked with mouse serum in PBS containing 2% BSA.Fusion proteins (25 μg/ml) were incubated in complete RPMI medium for 1h at 37° C. or at 4° C. To detect bound proteins, the cells wereincubated with rabbit anti-EDN antibody or rabbit IgG, followed witha-rabbit IgG/FITC Ab incubation (Sigma) for 20 min each, and then fixedwith 1% paraformaldehyde. The binding-internalization was assessed viaflow cytometry on a FACScan (Becton Dickinson, Franklin Lakes, N.J.)using CellQuest software. For CCR4 expression, cells were incubated for45 min with FITC-conjugated anti-human CCR4 antibody (R&D) or withisotype-matched mouse IgG2b/FITC (R&D).

Example 1 Chemokine Fusion Proteins Kill Tumor Cells

Chemokine fusion proteins have been constructed with following toxicmoieties (i) EDN, a human pancreatic RNase, eosonophil-derivedneurotoxin, a member of the pancreatic RNase superfamily, previouslyshown to be potent cytotoxins for tumor cells after antibody targeting;(ii) human angiogenin, a member of the ribonuclease (RNase); or (iii) atruncated form of Pseudomonas exotoxin (PE38). The chemokines utilizedin the fusion constructs were selected based on their ability to targetCCR4 (TARC/CCL17 and MDC/CCL22) and CCR8 (I-309/CCL1, vMIP-I and MC148).Thus, chemotoxin proteins (MC148-EDN, mMIP3α-EDN, TARC-EDN andTARC-PE38) or control EDN fusion with mutant MC148 (MC148D-EDN), whichcan not bind CCR8, or TARC fused with tumor antigen OFA-iLRP (TARC-OFA)were constructed and produced at a high degree of purity (>95%).Angiogenin was excluded from further considerations due to itsnon-specific killing activity. Chemokine fusion did not affect RNaseactivity of EDN or function of PE38. Importantly, EDN or PE38 containingchemotoxins were able to kill only cells expressing a respectivechemokine receptor, such as MC148-chemotoxins and TARC-chemotoxinskilled cells via binding CCR8 or CCR4, respectively. Control cells thatdid not express CCR8 or CCR4 were not affected by treatment with MC148-or TARC-chemotoxins. PE38 containing chemotoxins were more potent thanEDN ones, which required at least 5-7 days for optimal killing.PE38-based chemotoxins, particularly TARC-PE38, were able to efficientlykill human T lymphoblastoid CCRF-CEM cells (CEM) in vitro within 1-2days of treatment (IC₅₀ of 3-8 nM). Moreover, s.c. established CEMtumors were killed by intratumoral injections of TARC-PE38 leaving onlyscar tissues at the site of injections. While mock treatment or TARC-OFAinjections failed to protect mice, 100% of mice treated with TARC-PE38survived and did not have palpable tumor during the observed period.

Schema of constructs are shown in FIG. 1. The data presented hereinindicates that chemokines, such as MC148 (viral chemokine encoded by thepoxvirus molluscum contagiosum) or thymus and activation-regulatedchemokine (TARC), fused with a toxin moeity can specifically target andkill cells which express CCR8 and CCR4, respectively. The datademonstrate that MC148-EDN, or TARC-EDN, or TARC-PE38 chemotoxins, butnot toxin alone, could bind to respective chemokine receptors, such asCCR8 and CCR4, respectively (FIG. 2 and FIG. 3).

Treatment of CD4+ T cells with TARC-EDN or TARC-PE38 preferentiallyeliminated/killed CD4+/CD25+/CCR4+ cells (FIG. 4). This is a veryspecific process, since unlinked toxin alone (EDN) or control antigenfused with TARC failed to kill cells (FIG. 4 and FIG. 6). Reduction orelimination of Tregs correlated with enhanced proliferative responses ofanti-CD3 stimulated CD8+ cells (FIG. 5). Moreover, chemotoxin treatmentinhibited Treg-mediated suppression of CD8+ T cells (FIG. 5). Moreover,chemotoxin treatment drastically augmented T cell responses, such asmixed lymphocyte reaction.

Moreover, chemotoxins efficiently killed CEM tumor cells both in vitroand in vivo in NOD-Scid mice (FIG. 6 and FIG. 7) suggesting thatTARC-based chemotoxins might be utilized for the therapy of T-cellmalignancies, such as cutaneous T-cell leukemia. Potency of chemotoxinsis very high: only ng/ml quantities required to eliminate 50-80% cells(FIG. 6).

Example 2 Toxins Delivered Via Chemokine Receptors, Chemotoxins,Specifically Kill Tumor Cells

Cell Lines and Mice. Human acute T-lymphoblastic leukemia cell linesCCRF-CEM (CCL-119, CEM), and MOLT-4 (CRL-1582), and human embryonickidney 293 cells (HEK-293) were purchased from ATCC (Manassas, Va.).HEK-293 cells stably transfected with human CCR8 (HEK/CCR8) were a giftfrom Dr. Zack Howard (Science Applications International Corporation,Frederick, Md.). HEK-293 cells were cultured in Dulbecco's modifiedEagle's medium (Invitrogen Corporation, Carlsbad, Calif.) containing 10%fetal bovine serum (FBS). The same medium but with 400 μg/ml of G418(Sigma-Aldrich, Inc., Saint Louis, Mo.) was used to maintain HEK/CCR8cells. CEM and MOLT-4 cells were cultured in a standard RPMI medium 1640(Invitrogen Corporation) supplemented with 10% FBS. Female 6-8 weeks oldNOD/LtSz-scid/scid (NOD/SCID) mice were purchased from The JacksonLaboratory (Bar Harbor, Me.) and kept under pathogen-free conditions atthe National Institute on Aging animal facility, Baltimore, Md. Animalcare was provided in accordance with the procedures outlined in theGuide for the Care and Use of Laboratory Animals (NIH Publication No.86-23, 1985).

Plasmid Constructs. Schema of constructs is shown in FIG. 1. Maturesequence for TARC/CCL17 was cloned using RT/PCR from human thymus RNAusing the following pairs of primers PRhTARCM-1 (ATCACCATG GCA CGA GGGACC AAC GTG GGC CGG GAG T (SEQ ID NO:11)) and PRhTARC-GC-R3 (ATA CTC GAGGCT ACC CCC ACC GCC AGA GCC ACC ACC ACC AGA CCT CTC AAGGCTTTGCAGGTA (SEQID NO:12)). Eosinophil-derived neurotoxin (EDN) was recloned using PCRwith primers: PREDN-1 (ATA CTC GAG GGT GGT GGT GGT TCT AAA CCG CCG CAGTTC ACT TGG GCT(SEQ ID NO:13)) and PREDN-R2 (CGC GGA TCC GAT GAT ACG GTCCAG ATG AAC CGG AAC (SEQ ID NO:14)) from the EDN expressing plasmiddescribed. Similarly, angiogenin (ANG) was PCR recloned from anANG-expressing plasmid (Rybak S M et al. (1992) PNAS 89: 3165-3169)using primers PRAngi-1 (TATCATATGC TCGAGGGTGG CGGTGGAAGC CAGGATAACTCCAGGTACAC ACACTTCCT (SEQ ID NO:15)) and PRAngi-R1 (ACTGGATCCCGGACGACGGA AAATTGACTGA (SEQ ID NO:16)). Bacterial expression vectorswith CCR8 antagonist encoding mature sequence of MC148 (MC148) or mutantMC148 (MC148D, which could not bind CCR8 due to single a.a. residuereplacement) of Molluscum contagiosum virus have been describedpreviously 27. A truncated (38 kDa) form of Pseudomonas exotoxin (PE38)that does not have cell binding and internalization domain 28 wasrecloned from the pOND 21-2 plasmid (gift of Dr. Ira Pastan, NCI,Bethesda, Md.) using PCR with following primers: PRPE38-3 (ATAACCATG GAAGCT TCT GGA GGT CCC GAG GGC GGC AGC CTG GCC GCG CTGA(SEQ ID NO:17)) andPRPE38-R2 (TAT AGA TCT CTT CAG GTC CTC GCG CGG CGG TTT GCC GGG CTGGCT(SEQ ID NO:18)). To construct a chemotoxin expressing vector, PCRfragments encoding for mature sequence of human TARC were cut with NcoIand XhoI enzymes and ligated in frame with XhoI and BamHI cut fragmentsencoding for EDN (TARC-EDN) or ANG (TARC-ANG), and inserted into thebacterial expression plasmid pET11 opened with NcoI and BamHI (Novagen,Madison, Wis.) that was modified to contain also c-myc and six His tags(SEQ ID NO: 25). To construct EDN fusions with wild-type MC148 or mutantMC148D, XhoI and BamHI-cut END fragments were cloned into the XhoI andBamHI-opened pMC148-VL315 and pMC148D-VL315 plasmids. To constructchemotoxins with PE-38, PE38 fragment with Hind III (cut and blunted)and BglII ends were ligated into expression vectors for TARC or MC148opened with XhoI (blunted) and BamHI (TARC-PE38 and MC148-PE38,respectively). The following control constructs were used: plasmidsexpressing MC148 or TARC fused with irrelevant tumor antigen like murineembryonic antigen OFA-iLRP (TARC-OFA) or plasmacytoma MOPC315-derivedVL315 (MC148-VL315). A spacer fragment was inserted betweenchemoattractant and fusion moieties to enable proper folding of theprotein. All constructs were verified by the DNA sequencing (Keck DNASequencing, Yale University, New Haven, Conn.).

Protein Production and Purification. Chemotoxin plasmid—containing BL21(DE3) E. coli cells (Stratagene, La Jolla, Calif.) were grown at 30° C.in 2×YT medium supplemented with 100 μg/ml each ampicillin andcarbenicillin and 1% dextrose. Chemotoxin production was induced for 6hours at 37° C. with 1 mM isopropyl-Beta-D-thiogalactopyranoside (IPTG)in the same growth medium except with 0.3% dextrose. Recombinantproteins were purified and refolded from bacterial inclusion bodies asdescribed previously³⁰. The integrity and purity of recombinant proteinswere on average more than 90%, as tested by SDS-PAGE under reducingconditions and verified by Western blot hybridization with monoclonal9E10 anti-c-myc antibody (Sigma) or with rabbit polyclonal anti-EDNantibody (use ref 41—you cannot buy this).

RNase Activity Assay. RNase activity was determined at 37° C. bymonitoring the formation of perchloric acid-soluble nucleotides asdescribed (ref 41). Each assay was repeated twice and the data pooled.

Chemokine Receptor Binding-Internalization Assays. Expression of CCR4and CCR8 were tested using corresponding fluorescein isothiocyanate(FITC)-conjugated anti-human CCR4 and CCR8 antibodies (R&D systems)according to manufacturer instructions. The ligandbinding-internalization assays were performed with 1×10⁵ cells blockedwith 10% mouse serum in PBS containing 2% bovine serum albumin (PBS-B).Proteins (50 μg/ml) were incubated in cell culture medium for 1 h at 37°C. or at 4° C., and, after extensive washings with PBS-B, incubated witheither anti-c-myc monoclonal antibody (Ab) (1:100 dilution, 9E10) oranti-EDN polyclonal rabbit Ab (1:100 dilution). Then cells wereincubated with respective secondary Abs conjugated with FITC, such asanti-mouse-IgG-FITC (Jackson ImmunoResearch Laboratory, Bar Harbor, Me.)and anti-rabbit IgG-FITC (Sigma) Abs. The binding/internalization wasassessed by flow cytometry on a FACScan (Becton Dickinson, FranklinLakes, N.J.) using CellQuest software. Alternatively, chemotoxin bindingand internalization was assessed by fluorescent microscopy. Briefly,cells (1×10⁵) were treated for 1 hour at 37° C. or at 4° C. with 25μg/ml fusion proteins in RPMI-1640 containing 10% fetal bovine serum(FBS). Then cells were fixed with 3.7% formaldehyde and permeabilizedwith 0.2% Triton X-100 (5 min) and incubated with mouse anti-c-myc Abfor 1 hour at 37° C., and for 30 min at 37° C. with goat anti-mouse IgGAb conjugated to Alexa Fluor 488 (Molecular Probes Inc, OR, USA). Imageswere acquired with a 40× objective on an Axiovert 200 microscope (CarlZeiss Vision GmbH, Munchen-Hallbergmoos, Germany) and using Axiovisonsoftware (Carl Zeiss Vision GmbH).

Chemotaxis assay. Human monocytes were isolated with the MACS CD14monocyte isolation kit (Miltenyi Biotec, Auburn, Calif.) from Ficolldensity gradient centrifuged peripheral blood mononuclear cells ofhealthy donors who provided signed informed consent. The purity was >95%as tested by flow cytometry. Chemotaxis was performed in AP48microchemotaxis chamber (Neuro Probe, Cabin John, Md.) followingmanufacturer's instructions.³¹ Briefly, lower wells were filled with 25μl complete RPMI medium containing titrated amounts of proteins or 1ng/ml of N-Formyl-Met-Leu-Phe peptide (a positive control, FMLP, Sigma).The top chamber was filled with 50 μl (1×10⁵) cells. Chambers wereseparated with 5 μm pore size membrane. Cells were incubated for 90minutes at 37° C. Non-migrated cells were gently removed and migratedcell were fixed and stained with haematoxylin/eosin and counted under100× magnification in 3 randomly chosen fields. Results are expressed asnumber of cells per microscopic field and represent mean±SD of 3 wells.

Cell viability assays. Cell viability was assessed using cellproliferation reagent WST-1 (Roche Applied Science, Indianapolis, Ind.).A minimum of three independent assays were performed in triplicate foreach cell type and protein. Cells (5×10⁴), plated in 96-well flat-bottomplates one day prior to the assay, were treated with titrated amounts ofproteins in complete RPMI medium supplemented with 1% FBS (for HEK-293cells or HEK/CCR8 cells) or 10% FBS (for CEM and MOLT-4 cells) andcultured for up to 7 days. Then, cell media were replaced with 10% WSTreagent and cell viability was assessed after 2-4 hours of incubation at37° C. Results are expressed in percentage of OD₄₅₀ values ofPBS-treated cells. For in vitro cell outgrowth assay, 1×10⁴ CEM cellswere plated in 96-well plate and treated with either PBS or 10 μg/ml ofproteins and number of viable cells was evaluated daily for three daysusing WST reagent. Results are expressed in percentage of OD₄₅₀ valuesof PBS-treated cells. Cell apoptosis was tested by staining them withAnnexin-V-Fluor Staining kit (Roche Applied Science) according tomanufacturer instructions and analyzed by flow cytometry.

CEM tumor growth in vivo. Six- to eight-week-old female NOD/SCID micewere challenged subcutaneously with 2×10⁷ CEM cells. Twelve days later,the mice were checked for the presence of palpable tumors and mice whichdid not develop tumors were excluded from the study. The remaining micewere randomized and intratumorally injected with 25 μg proteins in 100μl PBS (TARC-PE38 or TARC-OFA) daily for five days. Tumor size wasmeasured in perpendicular dimensions every other day and tumor surfacearea was calculated. Mice were euthanized at day 27 or when tumor areareached 400 mm² For histological analysis, tumor was fixed in 10%formalin and embedded in paraffin. Paraffin slides were stained withhematoxilin and eosin and analyzed with light microscopy. To study tumoroutgrowth, tumor cells from PBS and chemotoxin-treated mice wereisolated and cultured in vitro for 7 days. Then, these tumor-derivedcells were seeded in 96-well plates (5×10⁴ cells/well) and treated for 2days with TARC-PE38 or TARC-OFA, or PBS. Untreated parental CEM cellswere used as control. Cell viability was assessed with WST reagent asdescribed. Data were expressed as percentage of OD₄₅₀ values of controlcells.

Efficacy of systemic therapy with chemotoxin—At Day 0, NOD/SCID micewere injected i.v. (tail vein) with 1×10⁶ CEM cells (n=6). At Day 16mice were bled and mononuclear cells were isolated and stained withanti-human CD45-FITC Ab (R&D systems) and were analyzed by FACS. CEMcells were detected in peripheral blood of all mice (data not shown).Starting from Day 20, 10 μg of TARC-PE38 or CTACK-PE38 (controlchemotoxin) in 100 μl PBS were injected I.V. once a day for 4 days. Inour in vitro killing assays CTACK-PE38 did not significantly reduce theviability of CEM cells at the doses up to 25 μg/ml (data not shown). AtDay 25, mice were culled and spleens were removed. Some splenocytes werestained with anti-human CD45-FITC Ab and were analyzed by FACS toevaluate the percentage of spleen-infiltrating tumor cells. Ten millionsplenocytes were cultured in RPMI supplemented with 10% FBS for 10 daysand, thereafter, were counted and analyzed for human CD45 and human CCR4expression as markers of CEM cells.

Statistical analysis. All data are expressed as means±SD. Data wereanalyzed using a computer-based software system (StatView 5.0.1., SASInstitute Inc., Cary, N.C.). Differences were tested by analysis ofvariance followed by post hoc Scheffe's F-test.

Results

Chemokines Fused with Toxic Moieties Bind Respective Chemokine Receptorsand are Internalized to the Cytosol. Chemokines MC148 and TARC/CCL17were genetically fused with RNases, human eosinophilic RNase, EDN(MC148-EDN and TARC-EDN, respectively, FIG. 1) and human angiogenin(ANG, TARC-ANG, FIG. 1), or with a truncated form of Pseudomonasexotoxin PE38 (TARC-PE38, FIG. 1). The idea was that MC148- orTARC-chemotoxin would preferentially kill CCR8- and CCR4-expressingcells, respectively. Control constructs expressed MC148 or TARC fusedwith irrelevant non-toxic antigens, described previously (MC148-V1315and TARC-OFA, FIG. 1). In addition, toxins were also fused with a mutantMC148 that lost ability to bind CCR8 (MC148D-EDN, FIG. 1). All proteinswere in vitro refolded and purified to >95% purity from bacterialinclusion bodies as described in Biragyn, A., et al. ((1991). J.Immunol., 167: 6644-6653, 2001). Chemotoxins retained expectedfunctional properties of the fused moieties. MC148-EDN and TARC-EDN(FIG. 8) and ANG (data not shown) exhibited significant RNase activityin vitro, though several fold less than unlinked RNases, while no RNaseactivity was exhibited by controls (MC148-VL315, FIG. 8A). Moreover,TARC-EDN was able to attract human monocytes in a dose-dependent manner(FIG. 8B), suggesting that chemotoxins also retained the ability to bindto their respective chemokine receptor(-s). Indeed, MC148- andTARC-chemotoxins could specifically bind CCR8− or CCR4-expressingHEK/CCR8 and CEM cells, respectively (FIG. 8C,D), but not thereceptor-negative control HEK-293 (FIG. 8C) and MOLT-4 cells (data notshown, and see FIG. 10A). Importantly, while TARC-chemotoxin was readilydetected on the surface of CEM cells incubated at 4° C. (wheninternalization of ligand-bound receptors was sequestered), it wasinternalized within few minutes after incubation of cells at 37° C.(TARC-EDN, left panel, FIG. 8D) and found in the cell cytosol(TARC-PE38, right panel, FIG. 8E). The internalization was through aspecific chemokine receptor as TARC-EDN down regulated surfaceexpression of CCR4, when incubated at 37° C. (FIG. 8D), but not at 4° C.No CCR4 binding or modulation was detected in cells treated with EDNalone (FIG. 8D). Thus, chemotoxins retained functional properties of thefused moieties and were internalized to the cell cytosol utilizing theirrespective chemokine receptors.

Properties of RNase-based chemotoxins. EDN-chemotoxin would presumablyrequire a delivery to the cell cytosol or nucleus in order to be able todegrade cellular RNA and induce cell death. To test this, HEK/CCR8 cellswere incubated with titrated amounts of MC148-EDN, or control proteinssuch as EDN alone and MC148-VL315. As shown in FIG. 9A, HEK/CCR8 cellswere specifically killed only when incubated with MC148-EDN, but notwith EDN alone or control MC148-VL315 protein. The cytotoxicity wasspecific to CCR8-expressing cells as MC148-EDN failed to killCCR8-negative parental HEK-293 cells (data not shown). Similarly, EDNfused with another chemokine TARC (TARC-EDN), but not EDN alone, wasalso able to kill CCR4-expressing CEM cells (FIG. 9B). In contrast,TARC-ANG exhibited cytotoxicity, regardless of CCR4 expression (data notshown), thus this fusion was not studied further. Taken together, thesedata suggest that EDN was successfully rendered cytotoxic, if deliveredto the cell cytosol as fusions with chemokines, presumably acting viadegradation of intracellular RNA. However, despite the fact thatrelatively low concentrations of EDN-chemotoxin (30-100 μg/mL, FIG. 9C)induced cell death, it was only detectable after prolonged incubation5-8 days (FIG. 9B).

Chemokine receptor expressing tumor cells are selectively andefficiently killed by chemotoxins in vitro. Bacterial toxins such as theexotoxin of Pseudomonas aeruginosa were reported to kill mammalian cellswithin a short period of time by inhibiting protein synthesis via theADP-ribosylation of elongation initiation factor 2 (Allured, V. S., etal. (1986) Proc. Natl. Acad. Sci. U.S.A, 83: 1320-1324 and Li, M., et al(1995) Proc. Natl. Acad. Sci. U.S.A, 92: 9308-9312). Indeed, TARC-PE38exhibited faster dynamics of cell killing than TARC-EDN. Overnightincubation with TARC-PE38 induced a marked increase in the apoptosis ofCEM cells (FIGS. 9C and D), and after two days of incubation, resultedin 80% dead cells (FIGS. 9C and E) Similar to the EDN-based chemotoxins,TARC-PE38 also required CCR4, since it only killed CCR4-expressing CEM,and not CCR4-negative MOLT-4 cells (FIG. 10A). Control formulationsTARC-OFA and TGFα-PE38 were not able to kill any cells, furthersupporting the requirement for chemokine receptor delivery to renderPE38 cytotoxic (FIG. 10B). TARC-PE38 exhibited high potency as itinduced significant killing at doses as low as 100 ng/ml (FIG. 10A), anda single overnight pretreatment with TARC-PE38 completely suppressedgrowth of fast growing CEM tumor cells in vitro (FIG. 10C). None of thecontrols used (TARC-OFA or EDN, FIG. 10) nor TARC-EDN affected thegrowth of CEM cells that multiplied 15-fold in three days.

TARC-PE38 Effectively Eradicates Established CCR4⁺ Cutaneous Tumor inMice. Patients with cutaneous T cell leukemia that express CCR4 have apoorer clinical outcome than CCR4⁻ cutaneous T cell lymphomas. Due tothe lack of murine models for cutaneous leukemia, we have established asubcutaneous human T-cell leukemia CEM model in NOD-SCID mice. Mice wereinjected in the flanks with CEM cells. At day 12 post-innoculation thetumor size reached 25-30 mm³ Mice were injected intratumorally with PBSor 25 μg of TARC-PE38, or TARC-OFA once daily for five consecutive days.Animals injected with PBS or the control TARC-OFA showed similar tumorgrowth and were sacrificed by day 27 when tumor size reachedapproximately 400 mm² (FIGS. 11A and 11E). In contrast, no measurabletumor was detected in mice treated with TARC-PE38, which was alsoassociated with the appearance necrotic lesions at the tumor challengesite (FIGS. 5b and 5e ). The data were supported by histologicalanalysis taken at 27 day after tumor challenge. Samples from micetreated with TARC-OFA consisted primarily of tumor cells (cells withlarge and dense nuclei, FIG. 11C) infiltrated in the dermis and spreadinto subcutaneous tissue. In comparison the majority of the TARC-PE38treated samples were free of tumor cells (FIG. 11D).

Tumor Relapse Due to Survival of Receptor-negative Cells in NOD-SCIDMice.

Despite the potency of the treatment and the fact that the TARC-PE38treatment eliminated any palpable signs of tumors in mice, tumoreventually relapsed and tumor cells could be detected at the margins ofthe necrotic area and surrounding skin by day 27 after tumor challenge.Question-there is a disconnect here-how can you show tumor freehistology and now take on the same samples and show tumor? However, thetumor cells had a different morphology than the initial tumor; cellswere smaller in size with less dense nuclei as compared to the originalor those from TARC-OFA-treated mice (data not shown). In addition, thetumor no longer responded to the TARC-PE38 treatment, and even higherdoses and multiple injections of the chemotoxin could not affect tumorgrowth (data not shown). Therefore, we hypothesized that these tumorsmight represent an outgrowth of resistant cells that had lost expressionof CCR4. To investigate, we cultured ex vivo cells removed from theserelapsed tumors or from PBS treated mice. Cells were then treated invitro with titrated doses of TARC-PE38 for two days. TARC-PE38 killed exvivo grown tumor cells from the PBS-treated mice as efficiently asparental in vitro cultured CEM cells (FIG. 12A). In contrast, cells fromthe TARC-PE38 treated and relapsed tumors were significantly lessaffected by TARC-PE38 (survival significance of p<0.001, FIG. 12A),indicating that these cells were less sensitive to the TARC-PE38. Toassess the mechanism of resistance, the parental in vitro cultured CEMcells were pretreated with PBS or 10 μg/ml of TARC-PE38, or TARC-OFAonce or for five days every other day. As we described, a singlepretreatment with TARC-PE38, but not with TARC-OFA or PBS, caused adramatic cell death yielding few viable cells (less then 5%, data notshown). The remaining cells were then cultured in complete mediumwithout chemotoxin for four weeks and analyzed for CCR4 expression.While the majority of the TARC-OFA- or PBS-treated cells expressed CCR4(97%, FIGS. 12B and C), pretreatment with TARC-PE38 for a single timesignificantly reduced proportion of CCR4-expressing cells (48%, p<0.001,FIGS. 12B and C). Moreover, when cells were pretreated with TARC-PE38five times and cultured for four weeks, the majority of cells wereCCR4-negative (78%, FIGS. 12B and C). These cells not only becameCCR4-negative, but also acquired resistance to subsequent treatmentswith TARC-PE38 (FIG. 12D). Control pretreatments with either PBS orTARC-OFA did not affect either the proportion of CCR4-expressing cells(FIGS. 12B and C), or sensitivity to TARC-PE38 (FIG. 12D). Overall,these data indicate that chemotoxin can be efficiently used foreradication of CCR4-positive cutaneous T cell tumors, that is oftenassociated with a poorer disease outcome. However, it will not eliminateCCR4-negative tumor variants and, thus, may lead to tumor relapse ofCCR4-negative escapees in immunosuppressed hosts (NOD-SCID mice).

Systemic Treatment with TARC-PE38 Depletes Spleen-infiltrating TumorCells in Mice—Systemic administration TARC-PE38 was also very effectivefor therapy of disseminated CEM cell growth in NOD/SCID mice (FIG. 13A).CEM cells express CD45 and in vitro cultured tumor cells were 100%positive for this marker (FIG. 13B). The percentage of humanCD45-positive cells was dramatically reduced in mice treated withTARC-PE38 compared with that in control chemotoxin-treated mice (FIG.13A). To confirm that cells-positive for human CD45 are indeed tumorcells, we cultured cells isolated from spleens for 10 days and evaluatedcell number and expression of CEM cell markers. The number of cellsexpanded from spleens of TARC—PE38—treated mice was more than 20-foldless compared to those from CTACK—PE38—treated mice (14.5±6.2×10⁶ vs.0.7±0.5×10⁶, respectively, P<0.05). While 100% of expanded cells in bothgroups were CD45-positive, CCR4 expression in cells cultured fromspleens of TARC-PE38—treated mice was about 3-fold higher compared tothose from CTACK-PE38—treated mice (35.2±6.2% vs. 12.1±2.5%,respectively, P<0.005, and FIG. 13B).

Example 3 Human Peripheral Blood T Regulatory Cells, Functionally MatureCCR4⁺Tregs and Immature-Type CCR4⁻Tregs, Regulate Effector T Cells

Regulatory CD4⁺ T cells (Tregs) are emerging as key controllers ofperipheral tolerance to self- and allo-antigens. Their dysfunction leadsto the spontaneous onset of autoimmune disorders or suppression ofimmune responses associated with a poor disease outcome in patients witha variety of malignancies. Characterization of Tregs has beencomplicated by the lack of unique markers, although Tregs are primarilydefined as CD4⁺ T cells that express CD25 (IL-2Rα), CTL-associatedantigen 4 (CTLA-4) and scurfin, a fork-head box P3 (FoxP3) gene product.These genes are expressed at a constitutively high level and suppressthe proliferation of activated T cells and dendritic cells. To date, atleast two subsets of human natural Tregs, memory-type CD25⁺CD4⁺ Tregsand natural naïve CD4⁺Tregs (nnTregs), are known to exist. In addition,a separate group of IL-10 producing murine effector-memory Tregs(T_(REM)) and human Tr1 cells, which acquire regulatory functions bypolarizing human CD25⁻ CD4⁺ T cells, have also been reported. Thesevarious Tregs differ in their homing patterns reflected by thedifferential expression of chemokine receptors. For example, CCR7 isexpressed by nnTregs directing their homing to secondary lymphoidorgans; CXCR4 is thought to participate in the retention of Tregs inbone marrow, CCR6 in the recruitment of T_(REM) into the skin, and CCR4is associated with Tregs that migrate into the periphery. However, thenature of CCR4⁺Tregs in human peripheral blood remains unresolved,particularly, considering the fact that less than 5% of circulating CD4⁺T cells may be Tregs (CD25⁺CD4⁺, FIG. 14A), while CCR4-expressing cellsaccount for 20% lymphocytes, which also include Th2-polarized CD4⁺ Tcells that also exert suppressive functions via production ofimmunosuppressive cytokines. Although the mechanism of T cell regulationis shown to require both IL-10 and TGFβ for some Tregs (Tr1) orperforin/granzyme- and cell contact-dependent process, the extent of theparticipation of immunosuppressive cytokines has not been elucidated.

The following experiments, through the use of chemotoxins, demonstratethat human peripheral blood Tregs consist of, at least, two distinctsubsets, memory-type CCR4⁺Tregs and naïve-type CCR4⁻ Tregs.

Materials and Methods

Plasmid Constructs

Cloning strategy for chemokine-fused antigens has been describedpreviously. Briefly, the mature sequence for human TARC was cloned asdescribed in Example 2. A truncated form of Pseudomonas exotoxin (PE38)that does not have cell binding and internalization domain was reclonedby PCR as described in Example 2. TARC was genetically fused in framewith PE38 (TARC-PE38) or with irrelevant tumor antigen OFA-iLRP(TARC-OFA) and expressed from bacterial expression vector pET11(Stratagene, La Jolla, Calif.). All constructs were verified by the DNAsequencing (Keck DNA Sequencing, Yale University, New Haven, Conn.).Production of chemokine-fused proteins from TARC-PE38 orTARC-OFA-containing BL21 (DE3) E. coli cells (Stratagene) has beenpreviously described previously. The integrity and purity (>90%) ofrecombinant proteins were tested by SDS-PAGE under reducing conditionsand verified by Western blot hybridization with monoclonal 9E10anti-c-myc Ab (Sigma).

Human Peripheral Blood Cell Isolation

Human peripheral blood samples were collected from healthy donors inaccordance with Human Subject Protocol #2003054 by the Health ApheresisUnit and the Clinical Core Laboratory of the National Institute on Aging(NIA). CD4⁺ cells were isolated by negative selection using human CD4subset column kit (R&D Systems Inc., Minneapolis, Minn.) from PBMCsafter Ficoll-Paque (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.)density gradient separation according to the manufacturers'instructions. CCR4⁺CD4⁺ or CCR4⁻CD4⁺ cells were selected as describedpreviously (Pappas, J. et al. (2006) Immunol Lett. 102:110-114).Briefly, 1×10⁸ CD4⁺ cells (in 900 μl of PBS containing 0.5% BSA and 2 mMEDTA were stained with 100 μl of anti-CCR4-FITC Ab (R &D Systems) for 45min at 4-8° C., and separated using anti-FITC microbeads (MiltenyiBiotec, Auburn, Calif.) and MS columns (Miltenyi Biotech). To achievethe highest purity, two consecutive MS column runs were used and CCR4⁻cells were further depleted using LD columns (Miltenyi Biotec). CD25⁺cells were selected from CD4⁺ cells or CCR4⁺CD4⁺ and CCR4⁻CD4⁺ fractionsusing anti-CD25 Ab-coated microbeads (Dynabeads CD25; DynalBiotech/Invitrogen Corp., Carlsbad, Calif.). Beads were removed fromcells using DETACHaBEAD CD4/CD8 reagent (Dynal Biotech/InvitrogenCorp.). Cell purity was determined by FACS which was in average 94-98%for CD25^(high)CCR4⁺CD4⁺ (CCR4⁺Tregs) and 83-89% forCD4⁺CD25^(high)CCR4⁻ cells (CCR4⁻ Tregs, a lower purity of CCR4⁻ Tregspresumably was due to slightly lower CD25 expression). CD8⁺ cells wereselected using human T cell CD8 subset column kit (R&D Systems).Monocyte/macrophage-enriched PBMCs were isolated by plastic adherenceand were irradiated with 4500 rad before use as APCs for MLR or feedercells.

Flow Cytometry Analysis

Expression of surface markers was detected by staining with theappropriate fluorochrome-conjugated Abs followed by FACS using a FACScanflow cytometer and Cell Quest Pro software (BD Biosciences). Thefollowing Abs used were from R&D Systems: anti-CCR4-FITC, anti-CD25-PE,and anti-CCR7-FITC. Anti-CLA-FITC, anti-CD45RA-PE, anti-CD62L-PE andanti-CD95L (FasL) Abs were from BD Pharmingen (Franklin Lakes, N.J.).Anti-Granzyme A-PE (BD Pharmingen) and anti-Granzyme B-PE (CaltagLaboratories, Burlingame, Calif.) Abs were used for intracellularstaining. Cells were fixed and permeabilized with Fix&Perm CellPermeabilization Kit (Caltag Laboratories). To study the effect ofactivation some cells were stimulated with MACSiBead particles-coupledwith anti-CD3/CD28/CD2 Abs (Human T cell activation/expansion kit,Myltenyi Biotec) according to manufacturer's instructions.

RNA Isolation and RT-PCR

Total RNA from flow-sorted fractions of CD4⁺ cells was isolated usingTrizol reagent (Invitrogen Corp.) and RNeasy kit (Qiagen). RT-PCR wasperformed with primers reported by others: human FoxP3,5′-GAAACAGCACATTCCCAGAGTTC-3′ (SEQ ID NO:19) and5′-ATGGCCCAGCGGATGAG-3′(SEQ ID NO:20); human glyceraldehyde-3-phosphatedehydrogenase, 5′-TGTGGAAGGGCTCATGA CCACAGTCCAT-3′ (SEQ ID NO:21) and5′-GCCTGCTTCACCACCTTCTT GATG-3′(SEQ ID NO:22).

Suppression Assays

To test Tregs suppression, the responder CD8⁺ T cells (5×10⁴ each) werelabeled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester(CFSE, CellTrace Cell Proliferation Kit, Molecular Probes/InvitrogenCorp.) for 10 minutes in PBS at 37° C. Then cells were cultured withautologous APCs (5×10⁴) and titrated amounts of Tregs for 4-5 days inthe presence of soluble anti-CD3 Ab (0.5 μg/ml) in 96-well plates. Atthe end of experiment cells were stained with anti-CD8-PE Ab stainingwere analyzed by FACS. CD8⁺CFSE^(low) cells were considered asproliferated cells. The results are expressed as percentage ofproliferation of CD8⁺ cells cultured in the absence of CD4⁺ cells. SomeTregs were treated for 1 hour with soluble anti-CD3 Ab (5 μg/ml) andanti-CD28 Ab (1 μg/ml), washed and were cultured for 3 days in thepresence of recombinant human IL-2 (100 U/ml). Alternatively, anti-CD3Ab (0.5 μg/ml) was present or absent in the culture medium throughoutthe experiment with CD8⁺ cells in MLR assay with allogeneic APCs. Toneutralize FasL, anti-human FasL/CD95L Ab (20 μg/ml, NOK-1, NA/LE, BDPharmingen) or isotype-matched control Ab (BD Pharmingen) were used.Tregs or control cells were pre-incubated with Abs for 1 hour at 37° C.before adding to responder cells.

Depletion of CCR4⁺ Cells Using TARC-Chemotoxin

Human peripheral blood CD4⁺ and CD8⁺ cells were treated with 10 μg/ml ofTARC-PE38 or TARC-OFA or with PBS for 2 days in complete RPMI mediumsupplemented with 10% FBS and 5% human AB serum. Expression of CCR4 andcell death were evaluated using anti-CCR4-FITC Ab and propidium iodide(PI, Roche Diagnostics Corp.). Cell viability of purified CD4⁺ cellfractions treated as described above was assessed using CellProliferation Reagent WST-1 (Roche Diagnostics Corp.).

Effects of CCR4⁺ Cell Depletion on CD4⁺ Cell Activation andProliferation in Response to Polyclonal Stimulation

After overnight treatment with 10 μg/ml with proteins, CD4⁺ cells werewashed and activated with soluble 5 μg/ml anti-CD3 (BD Pharmingen) and 1μg/ml anti-CD28 Abs (BD Pharmingen) for 3 days which was continued for 4more days in the presence of 10 U/ml of IL-2 (PeproTech Inc., RockyHill, N.J.). Then, secreted cytokines were measured using Multiplex(BioRad), and cells were stained with anti-CD25-PE and anti-CCR4-FITCAbs and analyzed by FACS. Uptake of 100 nM of 5-bromo-2′-deoxiurydine(BrdU) for 2 hours was used to test for cell proliferation (ColorimetricCell Proliferation ELISA, Roche Diagnostics Corp., Roche AppliedScience, Indianapolis, Ind.). To study a role of Th2-type non-Tregs,CD4⁺ cells, depleted from CD25⁺ cells, were overnight treated with 10μg/ml of TARC-PE38, or TARC-OFA, or PBS, and, after extensive washes inPBS, the cells were activated with plate-bound anti-CD3 Ab (0.5 μg/ml)and 1 μg/ml soluble anti-CD28 Ab. Then, after 3 days cultivation, 3×10⁶cells/ml were plated and cultured for 3-5 more days in the presence ofIL-2 (10 U/ml) to determine secreted cytokines.

Animals

All mice were bred and housed at the National Institute of Aging animalfacility, Baltimore, Md. Animal care was provided in accordance with theprocedures outlined in a Guide for the Care and Use of LaboratoryAnimals (NIH Publication No. 86-23, 1985). The TCR transgenic pmel-1(Vα1Vβ13 T cell receptor for H-2D^(b)-restricted mouse and human gp100epitope) mice have been described previously³⁶. C57BL/6 mice werepurchased from Jackson (The Jackson Laboratory, Bar Harbor, Me.).

Effect of CCR4⁺ Cell Depletion on Antigen-Specific T Cell Proliferation

The synthetic peptides (purity >99%, Peptide Technologies, Washington,D.C.) used are human-gp100₂₅₋₃₃ (KVPRNQDWL; SEQ ID NO:23) and MOPC-315Ig₉₁₋₁₀₁ (ALWFRNHFVFGGGTK; SEQ ID NO:24). Pmel mice were vaccinatedsubcutaneously twice at 3-wk intervals with 10 μg human gp100₂₅₋₃₃peptide emulsified in 100 μl incomplete Freund's adjuvant (IFA, Sigma).Three weeks after the second vaccination, splenocytes were harvested andwere treated with either PBS or 10 μg/ml TARC-PE38 or TARC-OFA for 24hours. After washings, splenocytes were cultured in complete RPMI mediumcontaining 10% FBS, 20 IU/ml recombinant human IL-2 and 1 μg/mlcorresponding peptide (gp100₂₅₋₃₃ and MOPC315, respectively) for 3-5days and tested on irradiated target cells (DCs from C57BL/6 mice)pulsed with either gp100₂₅₋₃₂ or MOPC315 peptides. DCs were prepared aspreviously described³⁰. Secreted IFNγ was measured after 24 hours ofincubation by ELISA. Proliferation of splenocytes was measured by BrdUincorporation after 5 days of culture.

For suppression assays, CD8⁺ and CD4⁺ cells were isolated from pmel orC57BL/6 splenocytes, respectively, using murine T cell subset columnkits (R&D Systems). CD4⁺ cells were overnight treated with 10 μg/mlTARC-PE38 or TARC-OFA, or PBS and, after washings, they were mixed at1:1 ratio with pmel CD8⁺ cells and stimulated with gp100₂₅₋₃₂peptide-pulsed irradiated APCs. Cell proliferation was evaluated after 5days by BrdU incorporation. Mouse CCR4⁺ expression was detected withgoat anti-mouse CCR4 Ab (Axxora LLC, San Diego, Calif.) followed bystaining with FITC-conjugated secondary Ab (Sigma).

Statistical Analysis

All data are representative of at least 2 experiments. For all graphsdata represent mean±SD of triplicates. Differences were tested usingStudent's t test or one-way ANOVA followed by post hoc Scheffe's F-test.A p value less than 0.05 was considered statistically significant.

Results

Two Distinct Subtypes of Tregs

In humans, CD25⁺CD4⁺ cells represent up to 5% of circulating peripheralblood CD4⁺ T cells that can be further divided into two distinctpopulations of cells, CCR4⁺CD25⁺ (3.2±0.5%) and CCR4⁻CD25⁺ (1.9±0.6%)(FIG. 14A). These two populations expressed FoxP3 mRNA (FIG. 14B) andsuppressed proliferation of autologous CD8⁺ (FIG. 14C) and CD4⁺ cells Tcells stimulated with anti-CD3 Ab. In contrast, CCR4⁻CD25^(−/low),CCR4⁺CD25^(−/low) CD4⁺ T cells did not express FoxP3 (FIG. 14B) norsuppress T cell proliferation (FIG. 14C). Thus, CCR4⁺CD25⁺ and CCR4⁻CD25⁺ cells exert regulatory activity and designated CCR4⁺Tregs andCCR4⁻Tregs, respectively. Moreover, the cells appear to differ in theirdifferentiation state. While CCR4⁺Tregs (>90%) did not express CD45RAindicating to their memory phenotype, most of CCR4⁻Tregs expressedCD45RA⁺ and belonged to naïve cells (FIG. 1d ). The cells presumablyhome to different organs, since CLA⁺, a skin-homing marker, wasexclusively expressed by CCR4⁺Tregs as was also reported by others Incontrast, CCR4⁻Tregs did not express CLA (FIG. 14D), but expressed CCR7and CD62L, markers for homing to secondary lymphoid organs. However,CD62L and CCR7 were also expressed by the majority (≥90%) or half ofCCR4⁺Tregs, respectively (FIG. 14D), suggesting that Tregs may actuallyconsist of at least three distinct subgroups, namely CCR4⁺CCR7⁻,CCR4⁺CCR7⁺, CCR4⁻CCR7⁺ (FIG. 14D).

CCR4⁺ and CCR4⁻ Tregs are Functionally Different Regulatory Cells.

Based on the expression of markers for non-activated naïve cells(CD45RA⁺) and LN homing (FIG. 14D), we have postulated that CCR4⁻Tregsrequire an initial activation or instruction to become suppressivecells. Traditional assays, typically performed in the extended presenceof anti-CD3 Ab, would also activate Tregs and would not functionallydiscriminate between these two Treg populations (See, for example, FIG.14C). To study this, we tested Tregs for suppression of CD8⁺ Tcell-mediated MLR in the absence or presence of anti-CD3 Ab. Asexpected, both CCR4⁻ and CCR4⁺Tregs suppressed MLR at almost identicalcellular concentrations (FIG. 15A) in the presence of anti-CD3 Ab.However, when anti-CD3 Ab was omitted from the assay, only CCR4⁺Tregsbut not CCR4⁻Tregs inhibited MLR (FIG. 15B). Thus, the Tregs consist oftwo functionally distinct populations, mature CCR4⁺Tregs that exhibitregulation independent of pre-activation, and an immature-type ofCCR4⁻Tregs that require an initial TCR signaling to become a fullyactive regulatory cell. However, CCR4⁻Tregs are pre-sensitized, as onlya brief (1 hour) pre-treatment with soluble anti-CD3 Ab was sufficientto activate their suppressive effects (data not shown).

CCR4⁺ and CCR4 Tregs Use Different Regulatory Pathways

It is widely accepted that Tregs require cell contact to exert theirsuppressive effects. In concordance, conditioned medium from activatedmixtures of Treg/non-Treg cells, that contained significant amounts ofIL-10 (FIG. 16A), failed to inhibit proliferation of non-Treg cells (MedCD25⁺CCR4⁺, FIG. 16B). Recent reports have revealed that Tregs utilizeboth granzymes A and B (GZ-A and GZ-B) in contact-mediated cellsuppression. However, no expression of any GZ-A in Tregs was detectedregardless of CD3/CD28 Abs activation (data not shown). Similarly, nosignificant GZ-B expression was detected in non-activated Tregs (datanot shown). In contrast, activation with bead-bound anti-CD3/CD28 Absinduced significant GZ-B expression in CCR4⁻Tregs and non-Tregs, butnone in CCR4⁺Tregs (FIG. 16C).

Thus, granzymes are presumably not used by CCR4⁺Tregs, but they mayemploy other contact-mediated suppressive mechanisms, such as Fas/FasL(CD95/CD95L) signaling, which plays a major role in the physiologicalregulation of T cell homeostasis. To test this, expression of FasL/CD95Lwas assessed on these cells. Resting CD4⁺ T cells are reported not toexpress FasL, although it was shown to appear within 8 hours ofstimulation. Moreover, FasLexpression was not detected on the surface offreshly isolated Tregs (data not shown). Similarly, no FasL wasexpressed on CCR4⁻Tregs (FIG. 16D) or non-Tregs (both CCR4⁺ and CCR4⁻populations, data not shown). In contrast, significant levels of FasLwere detected exclusively on CCR4⁺Tregs after a brief in vitro culturewithout any stimuli (FIG. 16D). Moreover, upon activation withbead-bound anti-CD3/CD28 Abs, CCR4⁻Tregs also expressed significantlevels of FasL, but predominantly in the CCR4⁻Tregs that were convertedto CCR4⁺ (FIG. 16E). Thus, CCR4⁻Tregs presumably contain precursor poolsthat require activation not only to become CCR4⁺Tregs, but also toutilize FasL. To demonstrate the functional role of FasL, Tregssuppression assay was performed in the presence of FasL neutralizing Ab.As shown in FIG. 16F, incubation with neutralizing anti-FasL Ab, but notcontrol Ab, reversed the suppressive activities of both CCR4⁺Tregs andCCR4⁻Tregs significantly augmenting proliferation of CD8⁺ T cellsinduced with anti-CD3 Ab. Therefore, these data clearly indicate thatthe need for the contact-dependent regulation exerted by Tregs can be atleast in part explained by utilization of FasL signaling pathways.

CCR4⁺CD4⁺ Non-Treg Cells Control Th1-Polarization, while CCR4⁺TregsRegulate Proliferation of T Cells.

In human peripheral blood, expression of CCR4 was detect on two types ofcells, Tregs and Th2-type CD4⁺ non-Treg cells that may be potentiallyimmunomodulatory expressing the immunosuppressive cytokine IL-10.Therefore, to assess their regulatory functions, a specific and directmethod that depletes CCR4-expressing cells utilizing chemotoxin, arecombinant protein expressing TARC/CCL17 fused with a truncatedexotoxin PE38 (TARC-PE38) was developed. TARC-PE38, but not control TARCfused with an irrelevant tumor antigen (TARC-OFA), specifically killedthe CCR4⁺ T cells at nM concentrations (FIG. 17A). Purified CD4⁺ T cellswere pretreated overnight with TARC-PE38, or with TARC-OFA, or mocktreated with PBS; and, after several washing steps, they were stimulatedwith soluble anti-CD3/CD28 Abs. The magnitude of proliferation (P<0.01,FIG. 17B) was significantly augmented in cells pretreated withTARC-PE38, but not with TARC-OFA or PBS. Moreover, in contrast toTARC-OFA or PBS, the TARC-PE38 pretreatment also induced significantlyhigher IFNγ (p<0.01, FIG. 17C) and lower IL-10 and IL-5 (data not shown)secretions. Given that TARC-PE38 specifically and selectively killedCCR4⁺ T cells, these data suggest that CCR4⁺ cells kept transientlysuppressed the proliferation and Th1-polarization of CCR4⁻ cells. Insupport of this conclusion, purified/sorted CCR4⁻CD4⁺ T cells alone (<5%contaminating CCR4⁺ cells, CD4⁺CCR4⁻, FIG. 17B,C) demonstrated a similarlevels of proliferation and IFNγ production as T cells treated withTARC-PE38, while highly purified CCR4⁺CD4⁺ T cells proliferated poorlyand failed to secrete IFNγ (CD4⁺CCR4⁺, FIG. 17B,C). Thus, CCR4⁺ T cellsare primary cells that control Th1-polarization of CCR4⁻CD4⁺ T cells.

To further dissect roles of CCR4⁺ Tregs and CCR4⁺ non-Tregs, CD4⁺ Tcells were thoroughly depleted of CD25-expressing cells (Tregs) usinganti-CD25 Ab-coupled beads. The remaining non-Treg CD4⁺ cells (≥95%pure) proliferated well and did not inhibit the proliferation of other Tcells (see CD25⁻CCR4⁺, FIG. 14A), indicating to Tregs as a primaryregulator of T cell proliferation. The CD25-depleted cells weresubsequently pretreated with TARC-PE38, or TARC-OFA, or PBS, after whichthe cells were stimulated with anti-CD3/CD28 Abs. The cells pretreatedwith TARC-PE38 demonstrated dramatically reduced expression of IL-10(FIG. 17D) and other Th2-type cytokines such as IL-4 and IL-5, butsignificantly increased levels of IFNγ production (FIG. 17E). Incontrast, the cells pretreated with PBS or TARC-OFA producedsignificantly higher levels of IL-10 (FIG. 17D) and IL-4 and IL-5, andless IFNγ (FIG. 17E). Taken together, these data suggest that, whilecell proliferation is primarily regulated by CCR4⁺Tregs via cell-contactand FasL-dependent process, Th1 polarization is controlled by non-TregCCR4⁺ Th2 type cells presumably through the action of immunomodulatorycytokines IL-10 and IL-4.

Practical Implications: Depletion of Murine CCR4⁺ T Cells AugmentsAntigen Specific CD8⁺ T Cell Responses

Tregs have also been reported to suppress responses of tumorantigen-specific CD8⁺ T cells. Initial experiments indicated that only afraction of CD4⁺ T cells in naïve C57BL/6 mouse peripheral blood andspleen expressed CCR4 (4.2±0.5% and 2.6±0.4%, respectively). These cellspresumably also include Tregs, as specific proliferation (FIG. 18A) andIFNγ production of CD8⁺ T cells from TCR transgenic pmel mice tomelanoma-derived gp100₂₅₋₃₂ peptide was significantly suppressed uponthe addition of MHC-matched spleen-derived CD4⁺ T cells, regardless ofwhether CD4⁺ T cells were initially treated with PBS or pretreated withTARC-OFA. In contrast, the proliferation could be completely restoredwhen the CD4⁺ T cells were initially pretreated with TARC-PE38 (FIG.18A). In addition, pmel splenocytes pretreated with TARC-PE38 yieldedsignificantly higher cell proliferation (FIG. 18B) and IFNγ production(FIG. 18C) when stimulated with the gp100₂₅₋₃₂ peptide, compared tocells pretreated with TARC-OFA or PBS (FIG. 18B,C). TARC-PE38 pretreatedcells also showed slightly increased (but significant) proliferativeresponse in general even when stimulated with control peptide (p<0.05,FIG. 18B). Control peptide stimulation failed to produce IFNγ (FIG.18C). Thus, these results demonstrate that murine spleen-derivedCCR4⁺CD4⁺ T cells, presumably Tregs, suppress antigen-specific CD8⁺ Tcell responses. This also indicates that the suppressive state of Tcells can be completely reversed by the depletion of CCR4⁺ T cells usingTARC-PE38.

Conclusion

The data presented in this example demonstrate that human peripheralblood contains several distinct populations of suppressive CD4⁺ T cells:two discrete populations of Tregs that regulate T cell proliferation,differentiated by expression of CCR4, and non-Treg CCR4⁺ T cells thatcontrol Th1-cell polarization. Moreover, the data indicate that FasL issignificantly expressed on the surface of CCR4⁺Tregs even upon a briefin vitro culture without any additional stimulation. In contrast,CCR4⁻Tregs only expressed FasL after stimulation with anti-CD3/CD28 Abs.Importantly, neutralizing anti-CD95L Ab significantly reversed theinhibitory effects of Tregs on the proliferation of CD8⁺ T cells, thoughinvolvement of additional suppressive mechanisms can not be ruled outsince the inhibition was only partial. Thus, these data indicate thatboth CCR4⁺Tregs and CCR4⁻Tregs utilize the FasL/Fas signaling pathway,although later requires an additional stimulation via TCR to expressFasL and exert regulation.

Taken together, we have characterized functionally distinct mature andimmature subtypes of Tregs circulating in human peripheral blood,distinguished by the expression of chemokine receptors, such as CCR4 andCCR7, and utilization of unique suppressive mechanisms. Therefore,utilizing chemotoxins that specifically kill CCR4⁺ T cells wedemonstrate that these cells also exert suppression of Th1-polarizationand T cell proliferation, and their depletion can significantly augmentantigen specific CD8⁺ T cell responses and production of Th1-typecytokines. Accordingly, chemotoxins will have significant clinicalimplications for treatment of autoimmune diseases, tumors and chronicinfections, which are shown to be controlled by Th2-type cytokine skewedcells and Tregs.

Example 4 Depletion of Treg Cells for Improvement of Vaccine-Induced TCell Response

The following experiment was performed to determine if TARC-chemotoxinswill specifically kill Treg cells. Since the systemic depletion of CD25⁺Treg cells can also induce harmful unpredicted autoimmunity, such asinduction of onset of gastritis, thyroidis, oophoritis or prostates. Weaimed to use chemotoxins locally to target only Tregs at the site ofvaccine expression or in local draining LN. The following datademonstrate that chemotoxin-mediated Treg depletion affects T cellresponses in vitro. TARC-chemotoxins efficiently depleted proportion ofCCR4⁺CD4⁺ cells within naïve T cell population after overnighttreatment. In contrast, proportion of CCR4-negative CD4⁺ or CD8⁺ T cellswere not affected by the treatment (both human or mouse cells, data notshown). Pre-treatment of human CD4⁺ T cells or purified CD4⁺CD25⁺ Tcells for two days with TARC-PE38, but not TARC-OFA or PBS, was able toeliminate their suppressive effects on proliferation of αCD3-inducedproliferation of CD8⁺ T cells. Similarly, Treg-suppressedantigen-specific proliferation of TCR transgenic CD8⁺ T cells from pmelmice, specific for H-2D^(b)-restricted mouse and human gp100₂₅₋₃₃epitope, was reversed by pretreatment of autologous CD4⁺ cells withTARC-PE38. Moreover, pretreatment with TARC-PE38 also significantlyaugments IFNγ production from pmel CD8⁺ T cells stimulated with APCspulsed with gp100₂₅₋₃₃ peptide. In contrast, mock PBS or TARC-OFAtreatments failed to augment either of the responses. Taken together,these data clearly demonstrate that chemotoxin-mediated depletion ofTregs improves antigen-specific CD8⁺ T cell responses.

Conclusion

The above-identified experiments confirm that unwanted cells can bedepleted as desired. The strategy is based on the fact that chemokinescan deliver antigens to the cytosol of cells expressing specificchemokine receptors. Chimeric proteins were produced, designatedchemotoxins, which consisted essentially of a chemokine fused with atoxic moiety. Only nM quantities of chemotoxins are sufficient tospecifically kill tumor cells expressing the proper chemokine receptors.Moreover, injections of TARC-chemotoxin into CEM tumors established inNOD-SCID mice elicit 100% tumor-free mice. Together, these resultssuggest that chemotoxins are useful in the treatment of human T cellmalignant diseases when a patient's immune system is severelyimmunocompromised by disease or age.

INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A pharmaceutical composition comprising a fusionmolecule comprising a chemokine receptor ligand and a toxin moietycoupled via a peptide linker, and a pharmaceutically acceptable carrier,wherein the linker has an amino acid sequence comprising: amino acids 74through 89 of SEQ ID NO: 2, amino acids 74 through 93 of SEQ ID NO: 4,amino acids 76 through 88 of SEQ ID NO: 6, amino acids 92 through 99 ofSEQ ID NO: 8, or amino acids 92 through 103 of SEQ ID NO: 10, whereinthe chemokine receptor ligand is a chemokine selected from the groupconsisting of MC148, I-309, MDC and vMIP-1.
 2. The pharmaceuticalcomposition of claim 1, wherein the chemokine receptor ligand isspecific for a chemokine receptor selected from the group consisting ofCXCR4, CCR1, CCR2, CCR4, CCR5, and CCR8.
 3. The pharmaceuticalcomposition of claim 1, wherein the toxin moiety is a proteinaceoustoxin, or fragment thereof.
 4. The pharmaceutical composition of claim3, wherein the proteinaceous toxin is eosinophil-derived RNase,Pseudomonas exotoxin, diphtheria toxin, or anthrax toxin.
 5. Thepharmaceutical composition of claim 1, wherein the toxin moiety is anon-proteinaceous toxin.
 6. The pharmaceutical composition of claim 5,wherein the non-proteinaceous toxin is selected from the groupconsisting of a maytansinoid, a calicheamicin or a taxane.
 7. Thepharmaceutical composition of claim 1, wherein the toxin moiety aminoacid sequence consists of amino acids 90 through 223 of SEQ ID NO: 2 oramino acids 94 through 435 of SEQ ID NO:
 4. 8. The pharmaceuticalcomposition of claim 7, wherein the fusion molecule comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO:
 7. 9. Thepharmaceutical composition of claim 7, wherein the fusion moleculeconsists essentially of an amino acid sequence that is at least 95%identical to SEQ ID NO:
 7. 10. A method for modulating an immuneresponse in a subject comprising: administering to the subject thepharmaceutical composition of claim 1, thereby modulating the immuneresponse, wherein modulating the immune response is selected from thegroup consisting of: increasing T cell mediated immune response in asubject; locally depleting T regulatory cells in a subject; inhibitingimmune suppression in a subject; modulating the suppressive effect ofCD4+CD25+ regulatory T cells of T effector cells in a subject;modulating the suppressive state of T cells in a subject; and shiftingan immune response from a Th2 response to a Th1 response in a subject.11. The method of claim 10, wherein the subject has cancer.
 12. Themethod of claim 11, wherein the cancer is a T cell malignancy.
 13. Themethod of claim 12, wherein the T cell malignancy is selected from thegroup consisting of cutaneous T cell leukemia and myeloma.
 14. Themethod of claim 10, wherein the subject has a chronic infection.
 15. Themethod of claim 14, wherein the chronic infection is selected from thegroup consisting of HIV infection, HCV infection, HBV infection and TBinfection.
 16. The method of claim 10, wherein the fusion molecule isadministered to the location of a tumor.
 17. The method of claim 10,further comprising administering to the subject a vaccine near thelocation of the administration of the fusion molecule.
 18. The method ofclaim 10, wherein the fusion molecule is administered locally.
 19. Themethod of claim 10, wherein the subject is being administered a vaccine.20. A pharmaceutical composition comprising a fusion molecule comprisinga chemokine receptor ligand and a toxin moiety coupled via a peptidelinker and a pharmaceutically acceptable carrier, wherein the chemokinereceptor ligand is TARC, I-309, MC148, or vMIP-1, and the toxin moietyamino acid sequence consists of amino acids 94 through 435 of SEQ ID NO:4, wherein the linker amino acid sequence comprises: amino acids 74through 89 of SEQ ID NO: 2, amino acids 74 through 93 of SEQ ID NO: 4,amino acids 76 through 88 of SEQ ID NO: 6, amino acids 92 through 99 ofSEQ ID NO: 8, or amino acids 92 through 103 of SEQ ID NO:
 10. 21. Thepharmaceutical composition of claim 20, wherein the chemokine receptorligand amino acid sequence consists of: amino acids 3 through 72 of SEQID NO: 2, amino acids 3 through 88 of SEQ ID NO: 6, or amino acids 3through 82 of SEQ ID NO:
 8. 22. The pharmaceutical composition of claim20, wherein the fusion molecule further comprises a c-Myc-His tag havingan amino acid sequence comprising amino acids 226 through 244 of SEQ IDNO: 2.